JP2001340089A - Scavenger receptor-like protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To isolate a gene specific to the nurse-like cell and clarify its function. SOLUTION: A new scavenger receptor-like protein is not only expressed highly in the nurse-like cell but also confirmed to be expressed in the vascular endothelial cell. Therefore, on the basis of the scavenger receptor-like protein, the screening of the compound useful not only in the diagnosis or treatment for RA but also in the diagnosis, prophylaxis or treatment for arterial sclerosis becomes possible.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a gene that is highly expressed in nurse-like cells and a protein encoded by the gene.

[0002]

Nurse cells are the first cells reported in the mouse thymus (Wekerle, H., et al., Nature, 283: 40).
2-404, 1980; Wekerle, H., et al., J. Exp.Med., 151: 9.
25-944, 1980). These cells are observed in vitro as adherent cells. Thymic nurse cells attached to a plastic petri dish can be relatively easily detached from the petri dish adhering surface by shaking the petri dish in the presence of EDTA, so that the adhesion is dependent on divalent metal ions such as Ca ²⁺ It is thought that it is brought by. Therefore, it has been speculated that integrin may be mainly involved in the adhesive activity of thymic nurse cells to the extracellular matrix (Takeuchi, E et.al., Arthritis
Rheum. 42: 221-228, 1999; Shimaoka, Y. et.al.J. Clin.
Invest., 102: 606-618,1998; Iwagami, S., Et.al.J.Imm
unol., 153: 2927, 1994).

In general, adhesion molecules are generated in various organs and tissues (D'Arcangelo, G., et al., Nature, 374: 719,199).
5), cell migration (Nakatsuka, K., et al., J. Rheumatol.,
24: 458, 1997) and that it is involved in physical processes such as the formation of inflammatory foci, wound healing, and metastasis of cancer cells (Hynes, RO, Cell, 69:11, 1992) has been reported. When thymic nurse cells are forcibly cultured in suspension with thymocytes, a complex of thymocytes and thymic nurse cells is formed. This phenomenon is emperipole
Called sis. Also, when co-cultured with thymocytes in the normal culture state, ie, in an adherent system, the thymic nurse cells “embrace” the thymocytes under the cytoplasm (pseudoemperipolesi
s) (Hiai, H., et al., J. Natl. Cancer Inst., 66: 713-72
2,1981) is observed. This is why they are called nurse-nurse-cells. Although the function of thymic nurse cells is still largely unknown, it is currently considered to have antigen-presenting ability and to be deeply involved in thymocyte clone selection and differentiation via MHC (Boyd, RL , et al., Immunol. Toda
y, 14: 445, 1993). In particular, thymic nurse cells may be involved in the acquisition of self-reactivity among several steps in thymic differentiation of T cells (Penninger, J., et a.
l., Eur. J. Immunol., 22:79, 1992; Rieker, T., et a
l., Eur. J. Immunol., 23: 904, 1993).

[0004] Cells similar to nurse cells are found in bone marrow (Shimao).
ka, Y., et al., J. Clin. Invest., 102: 606-618, 1998;
Tomita, T., et al., Rheumatology, 38: 854-863, 199
9) and skin (Nakagawa, S., et al., Eur. J. Immunol.,
23: 1705-1710, 1993: Iwagami, S., Et al., J. Immuno
l., 153: 2927, 1994), and are called nurse-like cells. Nurse-like cells are lymphocyte-penetrated beneath these cells in vitro (pseudoemperipolesi
s).

Further, the present inventors have isolated cells similar to nurse cells from synovial tissue of rheumatoid arthritis (hereinafter abbreviated as RA) patients. These cells were named "synovial nurse cells" because large mesenchymal cells harbor lymphocytes in vitro in primary cultures (heterologous cell populations) of RA synovial tissues in RA patients. (Toyosaki, T.,
et al., Arthritis Rheum., 41: 92-100, 1998; Shimao
ka, Y., et al., J. Clin. Invest., 102: 606-618, 1998;
Takeuchi, E., et al., Arthritis Rheum., 42: 221-228,
1999). In the present invention, the term “nurse-like cell” is used as a term that includes all of these nurse cells and nurse-like cells. There are many unknowns about the function of synovial cells, but synovial nurse cells hold T cells and B cells,
It can maintain long-term survival and maintain its proliferative ability against progenitor osteoclasts (Toyosaki, T., et al., Art
hritis Rheum., 41: 92-100, 1998; Shimaoka, Y., et al.,
J. Clin. Invest., 102: 606-618, 1998; Toyosaki-Maeda, T.,
Arthritis Rheum., 42: S155, 1999).

[0006] In the culture of nurse cells alone,
Alternatively, it has been reported that nurse cells produce various cytokines in a co-culture system with B cells (Tak
euchi, E. et.al., Arthristis Rheum., 42: 221-228, 1999;
Shimaoka, Y., et al., J. Clin. Invest., 102: 606-618,199
8). As cytokines produced by nurse cells, IL-1, IL-6, IL-8, G-CSF, GM-CSF, or TNF-
α and the like are known. These cytokines are all inflammatory cytokines. Synovial nurse cells also have an antigen-presenting function, similar to thymic nurse cells, and have an autologous mixed lymphocyte reaction.
ion: AMLR) (Tomas,
R., et al., J. Immunol., 152: 2613, 1994).

[0007] Synovial nurse cells are also considered to be involved in bone destruction of joints by inducing differentiation and activation of osteoclasts (Toyosaki-Maeda, T., Arth).
ritis Rheum., 42: S155, 1999). In other words, in the case of an inflammation such as RA, there is a possibility that the nurse cells and the blood cells interact to cause an inflammatory reaction. Therefore, it is important to elucidate the function of synovial nurse cells in elucidating the etiology and pathology of autoimmune symptoms in RA. Furthermore, it is essential to clarify the role of nurse cells in abnormal immune responses that cause various diseases, not limited to RA, in considering their prevention and treatment. In addition, if nurse cells are involved in the differentiation and activation of osteoclasts, the function of osteoclasts can be suppressed by inhibiting their functions, and control of bone destruction can be realized.

The present inventors have previously proposed NSR1 (Nurse cell Scavenger-like R
eceptor 1) has been isolated (mouse nurse cell receptor: JP-A-2000-236882; human nurse cell receptor: JP-A-2000-308492). NSR1 is a gene isolated by a subtraction method using mouse nurse cells (B6TNC) and mouse fibroblasts (L929). NSR1 was found to be homologous to SREC, one of the scavenger receptors described below. However, there is no report on the relationship between a nurse-like cell line existing in the synovium and each tissue and various diseases. Therefore, it is desired to isolate a gene specifically found in a nurse-like cell line present in the synovium and various tissues, analyze its function, and further clarify the relationship with a disease.

On the other hand, it has been clarified that a scavenger receptor is deeply involved in atherosclerosis. Macrophages infiltrating the vascular endothelium take up the oxidized low-density lipoprotein (oxidized LDL) via the scavenger receptor without saturating. As a result, macrophages become foam cells that have accumulated a large amount of oil droplets composed of cholesterol esters. The foam cells are expected to eventually develop into atherosclerotic lesions. Therefore, it is considered that the progression of arteriosclerosis can be suppressed by reducing the oxidized denatured product of serum lipid, that is, oxidized LDL and inhibiting the activity of scavenger receptor.

[0010] The first reported scavenger receptors were the bovine macrophage scavenger receptor types I and II (SRA-I and SRA-II) (Kodam).
a T. et al., Nature 343: 531-535, 1990). Then, MSR-I
II, MARCO (macrophage receptor with a collagenous
structure; Eloma Q., et al., Cell, 80: 603-609, 1995)
Scavenger receptors such as so-called class A type having a collagen-like structure and class B and C types having a structure different from class A have been reported. At present, eight macrophage scavenger receptors are known.

Furthermore, a scavenger receptor LOX-1 having oxidized LDL as a ligand has been isolated not only from macrophages but also from bovine arterial endothelial cells (Sawamura T., et al.).
al., Nature 1997, 386: 73-77). It has been suggested that LOX-1 acts in a direction that exacerbates vascular inflammatory symptoms. As a scavenger receptor derived from vascular endothelial cells, SREC (scavenger receptor expressed by end
otherial cells) are also known. SREC was cloned from human umbilical vein endothelial cell cDNA using oxidized LDL as a ligand (Adachi H., et al., J Biol. Chem. 1997, 272: 3).
1217-31220). In SREC, five cDNAs from SREC-1 to SREC-5 have been reported by alternative splicing. However, much is unknown about the function of SREC on the vascular endothelium. In any case, a scavenger receptor molecule expressed on vascular endothelial cells, which is a lesion site of arteriosclerosis, is expected to be closely related to the disease state. Therefore, scavenger receptors expressed in vascular endothelial cells can be useful proteins as targets for treatment or prevention of arteriosclerosis.

[0012]

An object of the present invention is to isolate a novel gene expressed in a nurse-like cell. Alternatively, an object of the present invention is to provide a novel scavenger receptor-like protein and a gene encoding the same. Another object of the present invention is to provide a novel use based on the function of a gene expressed in a nurse-like cell.

[0013]

Means for Solving the Problems The present inventors applied a technique for isolating NSR1 from mouse nurse-like cells to humans, thereby making it possible to identify specific Nurse-like cell lines present in human synovium and various tissues. We thought that it would be possible to isolate specific genes. That is, it was considered that a gene specific to human nurse-like cells could be isolated by a subtraction method between human nurse-like cells (tester) and non-nurse cells (driver). Tester cD required for subtraction method
NA was prepared by utilizing a nurse cell line discovered by the present inventors. This cell line allowed the subtraction method to proceed effectively. Further, based on the expression of the gene thus isolated in endothelial cells and the results of analysis of its structural characteristics, it was found that this gene is a novel scavenger receptor-like protein,
The present invention has been completed. That is, the present invention relates to the following scavenger receptor-like proteins and uses thereof.

[1] A protein comprising the amino acid sequence of SEQ ID NO: 2. [2] In the amino acid sequence of SEQ ID NO: 2,
Alternatively, a protein consisting of an amino acid sequence in which several amino acids have been substituted, deleted, added and / or inserted, and which is functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 2. [3] a protein encoded by a DNA that hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 and functionally equivalent to a protein consisting of the amino acid sequence of SEQ ID NO: 2; [4] a DNA encoding any of the proteins of [1], 2 and 3; [5] the DNA of [4], which comprises the coding region of the nucleotide sequence of SEQ ID NO: 1; [6] A vector into which the DNA of [4] or 5 has been inserted. [7] The vector according to [6], which is used for gene therapy. [8] A transformant carrying the vector of [6].

[9] A method for producing any of the proteins according to [1], [2], and [3], comprising a step of culturing the transformant according to [8] and collecting an expression product. [10] any of the DNAs of [4] or [5],
Alternatively, a DNA that hybridizes with its complementary strand and has a length of at least 14 nucleotides. [11] any of the DNAs of [4] or [5],
Or antisense DNA against a part thereof. [12] An antibody against any one of the proteins according to [1], [2], and [3]. [13] the antibody of [12], wherein the antibody is a monoclonal antibody; [14] the antibody of [12] or [13], wherein the antibody is at least partially humanized; [15] The protein detection reagent according to [1], comprising the antibody according to any one of [12], [13], and [14]. [16] A hybridoma that produces the monoclonal antibody according to [13]. [17] A method for detecting the activity of a test compound that binds to the protein according to [1], comprising the following steps: a) a step of bringing a test compound into contact with the protein of [1] b) a step of detecting the binding between the protein and the test compound [18] the activity of binding to the protein is detected by the method of [17] A method for screening a compound having an activity of binding to the protein according to [1], comprising a step of selecting the test compound thus obtained. [19] A method for detecting the activity of a test compound as a ligand for the protein according to [1], comprising the following steps: a) a step of bringing a candidate compound for a ligand into contact with cells expressing the protein of [1]; b) a step of detecting uptake by endocytosis via the protein; [20] the method of [19] The method for screening a ligand for a protein according to [1], comprising a step of selecting a compound in which activity as a ligand for the protein is detected. [21] A method for detecting the activity of a test compound that inhibits the binding of the protein described in [1] to its ligand, comprising the following steps: a) a step of bringing the protein according to [1] into contact with a ligand in the presence of the test compound, or a ') a step of bringing the protein according to [1] into contact with the test compound and then contacting with the ligand; 22] the protein of [1] and a ligand thereof, comprising a step of selecting a compound in which the activity of inhibiting the binding of the protein of [1] to its ligand is detected by the method of [21]; A method for screening for a compound that inhibits binding to a compound. [23] A compound obtainable by the method according to any one of [18], [20], and [22]. [24] Use of a compound obtainable by the screening method according to [20] or [22] as an inhibitor of cancer metastasis. [25] A method for screening a compound useful as an inhibitor of cancer metastasis by the screening method according to [20] or [22]. [26] A transgenic non-human vertebrate in which the expression of the protein encoded by the DNA according to [4] has been modified or the modification can be induced. [27] The transgenic non-human vertebrate of [26], wherein the expression of the protein encoded by the DNA of [4] is suppressed or the knockout animal is capable of inducing the suppression. [28] the transgenic non-human vertebrate of [26] or [27], wherein the non-human vertebrate is a mouse; [29] A method for producing a transgenic non-human vertebrate, wherein the expression of the protein encoded by the DNA according to [4] is modified or the modification can be induced, comprising the following steps. (A) a step of introducing a vector having the DNA sequence according to [4], a DNA sequence derived from a genome encoding the DNA, or a partial DNA sequence thereof, into a fertilized vertebrate egg or embryonic stem cell; A) generating an individual from a fertilized egg or embryonic stem cells of step (a); and (c) introducing the introduced DNA.
Selecting an individual that holds.

[0015]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel scavenger receptor-like proteins. The scavenger receptor-like protein of the present invention is a nurse cell line (human Hs6
It is a protein encoded by a gene cloned by subtraction between 7) and non-nurse cells. The amino acid sequence of the human Hs67-derived scavenger receptor-like protein “# 414” isolated in the present invention is shown in SEQ ID NO: 2, and the nucleotide sequence of the “# 414” cDNA is shown in SEQ ID NO: 1. Human # 414 gene consists of 742 amino acid sequences
It has an ORF and has at one position a region mainly composed of highly hydrophobic amino acids.

Analysis of the putative structure of the human # 414 protein revealed that although the amino acid sequence homology was low,
SRA-I (class A-I type scavenger receptor) and structural features were found to be completely identical.
FIG. 4 shows the structural features of # 414. That is, first, a cytoplasmic region consisting of 37 amino acids is present in the N-terminal region, and a sequence (YXRF) having a hook structure considered to play an important function in endocytosis is present in that region. Then 22
A hydrophobic region (one-time transmembrane type) considered to be a transmembrane region composed of amino acids, a spacer region composed of 383 amino acids and a coiled coil-like region are found.
In the coiled coil-like region, there was a leucine zipper structure in which a hydrophobic amino acid such as leucine appeared every 7 amino acids. In addition, there was a collagen-like region consisting of 147 amino acids, and in this region, 49 repetitions of the GXX sequence were observed. In addition, a large number of basic amino acids lysine or arginine considered to be important for binding to the ligand were present at the X position of GXX. In addition, histidine essential for dissociation of the ligand in the endosome was also present.

On the other hand, there is a type-specific region consisting of 153 amino acids at the C-terminal. In this area, # 414 is the conventional SRA-
It was confirmed that it was not a cysteine-rich domain found in I, MARCO, etc., but a lectin-like domain (C-type lectin region) involved in sugar chain binding. Further, as shown in the Examples, it is considered that this C-type lectin domain is involved in a function of recognizing galactose / N-acetyl-galactosamine (Gal / GalNAc) and taking it into cells. The above results support that the scavenger receptor-like protein of the present invention is a protein belonging to the class A scavenger receptor family in which the C-terminal type-specific region has been replaced by a C-type lectin region. Lectins that have been discovered in mammals (animal lectins) have been reported to be mainly involved in cell adhesion and endocytosis and also have an important role in host defense against infectious diseases. I have.

Based on such structural characteristics, the protein of the present invention was identified as a scavenger receptor-like protein. That is, the scavenger receptor-like protein in the present invention can be defined as a protein having the following structural characteristics. Techniques for searching for these structural features by amino acid sequence motif search are known.・ Has a cytoplasmic region ・ A sequence involved in endocytosis (YXRF) is present in the intracellular region ・ Has a transmembrane region ・ In the extracellular collagen region, a sequence GXX (G is glycine, X Is any amino acid) Lysine (K) which is a basic amino acid in any of X
Alternatively, the amino acid sequence of human # 414 of the present invention and the known human scavenger receptor SRA-I are only 16% homologous and form clusters in which arginine (R) is present. No protein could be found. Therefore, # 414 is a novel protein.

When the expression of human # 414 mRNA was analyzed by Northern blotting, a strong signal of about 3 kb was detected in lung, heart, placenta, small intestine, large intestine, prostate, testis, and ovary. Weak expression was seen in the tissues (FIG. 6). This suggests that the human # 414 protein may function in various tissues in a living body. On the other hand, no signal was detected in spleen, peripheral blood leukocytes, or liver. Hs67 and HSN2 are nurse cells in cell lines
7. Since expression was confirmed in SK-LMS-1, human # 414 is presumed to be involved in the function of nurse cells (FIG. 5).
Further, since expression was observed in all three types of human vascular endothelial cells, it is expected that the protein of the present invention is involved in arteriosclerosis (FIG. 7).

Based on the above findings, the receptor function of human # 414 and the role of disease are considered. Cell uptake of antigen proteins for antigen presentation, (2) biological defense from bacteria and viruses, and (3) cell adhesion. Hereinafter, these functions will be described in detail, and further, the relation between these functions and diseases will be discussed.

[0021] The scavenger receptor is a term that refers to a protein that recognizes various substances such as a part of nucleic acids, sugar chains, and denatured proteins, and has an action of taking up and removing these into cells. Since the ligand-binding domain of macrophage scavenger receptor (SRA), which is a typical scavenger receptor, has the same structure as collagen, which is the most important component of the extracellular matrix, cleaning treatment of foreign substances and waste that easily adhere to collagen is performed. Is believed to be involved in # 414, which has a collagen structure similar to that of SRA, is thought to be involved in the treatment of foreign substances and waste products in nurse cells and endothelial cells. In addition, since nurse cells have the ability to present antigens, these foreign substances, waste products, especially some modified / denatured self antigens are taken up by endocytosis via # 414, and a part of them is presented to immune cells. (Antigen presentation). This effect is
It is expected to be involved in the pathological condition as an autoimmune disease in RA and the like. Furthermore, it has been reported that these denatured (oxidized) proteins have increased antigenicity and elicit an antigen-specific immune response (Mamula, M. et al., J. Biol. Chem.,
274: 22321-22327, 1999), and indeed, it has been shown that scavenger receptors play an important role in the uptake of these antigens (Abraham,
R. et al., J. Immunol., 158: 4029-4035, 1997; Nic
oletti, A. et al., Eur. J. Immunol., 29: 512-521,
1999; Bansal, P. et al., J. Immunol., 162: 4430-443
7, 1999).

It has been shown that the # 414 gene is highly expressed in cultured vascular endothelial cells. Also, since # 414 has a lectin domain in addition to a collagen domain, glycoproteins, especially its sugar chain recognition domain (CRD: Carbohydrate Recon
From the characteristics of gnition domain), it can be expected that galactose (Gal) specifically recognizes glycoproteins. Since most human glycoproteins have sialic acid added to the terminal of the sugar chain, asialoid (deacylated) glycoprotein from which sialic acid has been removed is considered as the ligand of # 414. On the other hand, it has been reported that asialylated (desialylated) glycoproteins are associated with diseases in which asialized IgG is increased in Sjogren's disease (Youinou, P. et al., J. Autoimmun.,
Similarly, in RA and systemic lupus erythematosus (SLE), the presence of immunoglobulins and serum proteins with abnormal sugar structures has also been reported in RA and systemic lupus erythematosus (SLE) (Yagev, 1993). H. et al., Clin. Exp. Imm
unol. 94: 452-458, 1993; Panzironi, C., et al., Bio.
Chem. Mol. Biol. Int. 43: 1305-1322, 1997). The causes of these abnormalities are considered to be glycosylation abnormalities or abnormalities in the processing system of proteins (including waste products) having abnormal sugar chains, and the # 414 gene is thought to be involved in the latter.

Furthermore, expression of MHC class II by IFN-γ treatment (Lian, RH et al., J. Immunol. 157: 8
64-873, 1996), and its expression is induced by lymphocyte adhesion independent of IFN-γ (Collinge, M. et al., J.
Immunol. 161: 1589-1593, 1998), it is predicted that vascular endothelial cells also have an antigen-presenting ability like nurse cells. Regarding whether endothelial cells recognize and bind glycoproteins specifically to galactose (Gal),
It has been reported that binding to galactosyl-BSA, mannosyl-BSA, and fucosyl-BSA was performed using an endothelial membrane. Experiments have also shown that galactosyl-BSA is incorporated (Kataoka M. et al., Blood 65: 1163-1171, 1985). As described above, the # 414 gene plays an important role in waste product processing or antigen presentation, in which endothelial cells recognize and bind glycoproteins specifically to Gal and take them into cells by endocytosis. It is quite possible.

Next, it is considered that the # 414 gene is also involved in biological defense. For example, a scavenger receptor SRA-I structurally similar to this gene, or SRA-I
Cells expressing I are gram-positive bacteria such as streptococci (Str.
eptococcus pyrogenes, Streptococcus agalactiae, St
aphylococcus aureus, Enterococcus hirae) (Dunne, DW, et al., Proc. N
atl.Acad.Sci.USA., 268: 3538-3545,1994; Krieger,
M., Curr. Opin. Lipidol., 8: 275-280, 1997). Also,
This binding is due to the Lipo on the surface of the negatively charged Gram-positive bacteria.
It is presumed to be due to the interaction between teichoic acid and the collagen region of SRA with a positive charge. The result is
This suggests that scavenger receptor-like protein # 414 may be involved in early defense in infectious diseases,
The lack of # 414 is expected to lead to susceptibility. Also,
Although the carbohydrate-binding specificity is different, it has been reported that mannose-binding lectin (MBL), which is homologous to # 414, is associated with the deficiency and susceptibility to infant infection (Supe
r, M. et al., Lancet 2: 1236-1239,1989; Summerfiel
d, JA et al., Lancet 345: 886-889, 1995; Luty, A.
J. et al., J. Infect.Dis. 178: 1221-1224, 1998; Mads
en, HO et al., Lancet 352: 959-960, 1998). Furthermore, it has been pointed out that MBL replacement therapy may be used to prevent pulmonary infections [Garred, P. et al., J. Clin. Invest.
104: 431-437, 1999).

On the other hand, dendritic cells (Dendritic cells) and Langerhans cells (L
angerhans cell) has a C-type lectin region
DEC-205 receptor (Jiang, W., et al., Nature, 375: 151, 1
995) and Langerin (Valladeau, J., et al., Immunity,
12:71, 2000) is known to be involved in antigen presentation.
Similarly, Mannan-binding p with C-type lectin structure
rotein (Ikeda, K., et al., J. Biol. Chem., 262: 7451,198
7) and Natural killer receptor protein 1 (Bezouska,
K., et al., Nature, 372: 150, 1994) and asialoglycoprotein receptor (Ozaki, K., et al., J. Biol. Chem., 267: 9229, 199).
2) are also involved in biological defense through recognition of pathogenic microorganisms and tumor cells. Some of these receptors, such as the antigen receptor of MHC-restricted immunocompetent cells, have been suggested to transmit stimuli into cells (Bezousk
a, K., et al., Nature, 372: 150, 1994). From such findings, it is expected that # 414 having a C-type lectin region is involved in antigen uptake, processing, or presentation. Therefore, for the diseases caused by these reduced functions, gene therapy by introducing the gene of the present invention becomes possible.

The gene of the present invention contains a C-type lectin region and is highly expressed in cultured vascular endothelial cells, and is also highly expressed in heart, lung, kidney, placenta, small intestine, etc. where vascular tissue has developed. Has a selectin-like function,
It may be involved in adhesion between endothelial cells and cells such as lymphocytes. Therefore, being involved in the presence of inflammatory cells at sites of inflammation such as atherosclerosis,
In other words, it is considered that it is involved in the occurrence and progress of inflammation such as arteriosclerosis. In addition, from its adhesive ability, it is also conceivable that it may play an important role in hematogenous metastasis of tumor cells. Indeed, it has been reported that Gal / GalNAc-specific C-type lectin on bone marrow endothelial cells plays an important role in hematogenous bone metastasis of prostate cancer, and therefore, its inhibitor is considered May inhibit metastasis (Ki
erszenbaum, AL.et al., Prostate 43: 175-183, 200
0). In mouse tumor models, lectin inhibition by D-galactose has been reported to inhibit liver metastasis (Isenberg J. et al., Anticancer Res. 17: 376).
7-3772, 1997).

Incidentally, it has been reported that the coiled coil region of SRA is involved in cell adhesion (Fraser, I. et al., Natur
e, 364: 343-346, 1993). Since # 414 has a similar structure, its cell adhesion ability may play an important role in lymphocyte recruitment. In fact, as described later, data showing cell adhesion activity was obtained for # 414 of the present invention. In addition, apoptotic cells have the plasma membrane component Phosphatidylserine (P
It has been reported that CD36, CD68, SR-BI, and LOX-1, including macrophage scavenger receptor SRA, bind to apoptotic cells and PS liposomes (Sambrano, GR et al., Proc. .Natl.Acad.Sc
i.USA., 92, 1396-1400, 1995; Platt, N. et al., Pro
c. Natl. Acad. Sci. USA., 93, 12456-12460, 1996; Fukas
awa, M. et al., Exp. Cell Res., 222, 246-250, 1996; M
urao, K. et al., J. Biol. Chem., 272, 17551-17557, 19
97; Oka, K. et al., Proc. Natl. Acad. Sci. USA., 95, 95
35-9540, 1998).

On the other hand, it has been reported that nurse cells not only maintain embraced lymphoid cells for a long period of time, but also that some cells are processed by apoptosis in nurse cells (Philp, D. et al.). ., Cell. Immuunol., 148: 30
1-315, 1993). It has also been reported that vascular endothelial cells also phagocytose aging red blood cells and apoptotic lymphocytes (Oka, K. et al., Proc. Natl. Acad.
Sci. USA, 95: 9535-9540, 1998). Since the # 414 gene is expressed in both of these cells, it is possible that these cells may be involved in the recognition of apoptotic cells via the # 414 receptor.

Therefore, the ligand of the scavenger receptor-like protein of the present invention is a very important substance for elucidating the scavenging mechanism via the receptor. Specifically, by clarifying the interaction between the receptor of the present invention and a ligand, it contributes to elucidating the action of nurse cells in eliminating and treating modified proteins, denatured (oxidized) proteins, or apoptotic cells. Furthermore, it also leads to clarification of the antigen presentation mechanism of nurse cells. Furthermore, by elucidating a series of scavenging mechanisms, foreign substances in the living body, various aging-related diseases such as arteriosclerosis caused by waste disposal, diabetic vascular disorder, cataract, Alzheimer's disease, etc.
Involvement in the etiology and pathology of autoimmune diseases including RA,
Furthermore, the regulatory mechanism of the immune network centered on nurse cells can be elucidated. Then, it is thought that it is possible to develop therapeutic drugs targeting proteins and genes that support the scavenging mechanism.

In addition, data showing the cell adhesion activity of the scavenger receptor-like protein of the present invention were obtained. Many factors involved in cell adhesion have been identified so far. In particular, the integrin family is deeply involved in adhesion between allogeneic and heterogeneous cells, such as leukocyte migration to inflammatory sites, migration of stem cells during ontogeny, and not only cell-cell but also cell-extracellular matrix. It is a group of molecules with various functions involved. It has been reported that such integrin-mediated adhesion does not show sufficient adhesive activity in the presence of immunosuppressive agents cyclosporin A and FK506. This phenomenon is attributed to the fact that the integrin is not activated by the action of the immunosuppressant, which causes the cell to become rounded and unable to adhere to the inner surface of the petri dish.

On the other hand, in the cells in which the present invention is forcibly expressed,
# 414 due to some resistance to cyclosporin
Is thought to exhibit cell adhesion activity by a different mechanism from integrins. As described above, the molecules involved in adhesion expressed in various cells including nurse cells are extremely diverse, which probably indicates the importance of adhesion molecules in vivo. It is presumed that it has been acquired in the course of evolution to guarantee a fail-safe mechanism of recognition. In fact, adhesion molecules are more than just adhesion between extracellular matrix and cells,
It is an important group of molecules that precisely regulates various responses of the living body, such as development and immune response, by moving intracellular signaling systems (Anderson, DC, et al., Annu. Rev. Med., 38:
174, 1987; Springer, TA, Nature, 346,: 425,1990;
Hynes, RO, Cell, 69:11, 1992). On the other hand, adhesion molecules are associated with cancer metastasis and invasion (Bliss, R. et al.
D. , Et al., Clin. Exp. Metastasis, 13: 173, 1995), I
Type allergy (Bentley, AM, et al., J. Allergy Clin.
Immunol., 92: 857, 1993; Gundel, RH, et al., J. Cli
n.Invest., 88: 1407, 1991; Nakajima, H., et al., J. Ex.
p.Med., 179: 1145, 1994), autoimmune diseases such as rheumatoid arthritis (Corkill, MM, et al., J. Rheumatol., 18: 145).
3, 1991; Kavanaugh, AF, et al., Arthritis Rheu
m., 37: 992, 1994). In line with these, nurse cells have a unique property of "embracing lymphocytes" related to immune function, so cell adhesion factors expressed in nurse cells can be used as target factors for the treatment of the above autoimmune diseases and allergies. Can be expected.

The scavenger receptor-like protein of the present invention can be purified from nurse cells, nurse-like cells, the above-mentioned tissues, and the like by a known method. Alternatively, it can be obtained as a recombinant using a gene encoding the amino acid sequence of the scavenger receptor-like protein of the present invention (for example, SEQ ID NO: 2). The gene encoding the amino acid sequence and a method for producing a recombinant using the gene will be specifically described later.

The novel protein scavenger receptor-like protein provided by the present invention has the following utility. First, the protein of the present invention or a fragment thereof can be used as an immunogen for obtaining the antibody. Alternatively, since the protein of the present invention specifically recognizes N-acetylgalactosamine, it is useful as a probe for analyzing a sugar chain structure. That is, for example, N-acetylgalactosamine present on the cell surface can be detected by contacting the cell with the labeled protein of the present invention. Cell surface galactose / N-acetylgalactosamine (Gal / GalNAc) is a group of cells that interact with nurse-like cells that express this receptor present in various tissues such as the thymus, synovium, skin, and vascular endothelium of RA patients. Useful for analyzing Also,
By analyzing the binding of this receptor to blood circulating desialylated glycoprotein, we can clarify the glycoprotein that this receptor binds and may take up into cells, and predict its involvement in disease as a result May be possible. Thus, galactose / N on the cell surface and on glycoproteins
-Acetylgalactosamine (Gal / GalNAc) is an important indicator in the analysis of cells that interact with nurse-like cells expressing this receptor, glycoproteins, and the like.

The scavenger receptor-like protein of the present invention is useful as a marker for human nurse-like cells because it is highly expressed in these cells. Since synovial nurse cells are characteristic cells in synovial tissue of RA patients, detection of synovial nurse cells in synovial tissue can be used as an index of RA. Therefore, the gene # 414 of the present invention and the protein encoded by this gene can be used as an index for synovial nurse cells for diagnosis of RA. In particular, the ligand-binding region of the protein of the present invention is useful as a marker for nurse cells because it is present on the cell surface and is easily detected. The sugar chain ligand binding region of the protein of the present invention is SEQ ID NO: 2
Is a region corresponding to 153 amino acid residues (positions 590 to 742) on the C-terminal side in the amino acid sequence shown in FIG. Further, it is expected that various bacteria and in vivo proteins will bind to the collagen region and the coiled coil region as well as the SRA. Synovial nurse cells may be closely related to the autoimmune symptoms of RA through the control of T cells and B cells, and its marker is an important tool for elucidating the pathology of RA. Becomes

When the scavenger receptor-like protein of the present invention supports the function of a nurse-like cell, a molecule that inhibits the function can be used for the prevention or treatment of RA. In the case of inflammation such as RA, nurse cells and blood cells may interact with each other to cause an inflammatory reaction. Therefore, the scavenger receptor-like protein of the present invention can be used as a target molecule for a medicament useful for treating an autoimmune disease including RA. Therefore, the scavenger receptor-like protein of the present invention is useful for screening a compound that can be used for prevention and / or treatment of RA. Nurse cells are also considered to be involved in the growth of blood cell stem cells and maintenance of self-renewal ability, and in the positive or negative selection of lymphocytes, and in the development of normal immune system and maintenance of memory cells. ing.

Nurse cells have also been found in primary and metastatic foci of cancer. Therefore, it is considered that by controlling the function of the scavenger receptor-like protein of the present invention, it is possible to regulate immune response and the like and to prevent or treat these diseases. The expression of the scavenger receptor-like protein of the present invention was detected not only in nurse-like cells but also in all vascular endothelial cell lines examined. Macrophage infiltration in vascular endothelial cells and uptake of oxidized LDL by endocytosis are said to be closely related to arteriosclerosis. Thus, expression of the scavenger receptor-like protein of the present invention in vascular endothelial cells suggests a relevance of this protein to arteriosclerosis. In addition, on vascular endothelial cells, adhesion molecules having a C-type lectin structure such as P-selectin and E-selectin are expressed, and monocytes / macrophages /
It has been suggested that it is a molecule that supports the aggregation of lymphocytes at focal sites (Asa, D., et al., J. Biol. Chem., 270: 1166).
2,1995).

On the other hand, since the present scavenger receptor-like protein also has a C-type lectin structure and has a cell adhesion ability, it may be involved in the aggregation of monocytes / macrophages / lymphocytes into atherosclerotic lesions. Can also be expected.
Therefore, the scavenger receptor-like protein of the present invention is useful for screening a compound that can be used for prevention and / or treatment of arteriosclerosis. Alternatively, the scavenger receptor-like protein of the present invention or its gene can be used as a diagnostic marker for arteriosclerosis.

Further, another lectin, E- or P-se
Molecules, such as lectin, involved in the binding between vascular endothelium and lymphocytes and cell adhesion have been reported. Recently, it has been reported that DC-SIGN, a receptor type C lectin belonging to the same gene family as # 414, is involved in cell adhesion between dendritic cells and lymphocytes (Geitenbeek TB, et al. C.
ell. 100.575-585, 2000), this gene is thought to be involved in adhesion between dendritic cells and lymphocytes. Since # 414 is expressed in vascular endothelial cells and # 414Lec-AP binds to lymphocytes as shown in FIG. 15, the # 414 gene is associated with vascular endothelial cells and lymphocytes (some T and B cells). ) May be involved in cell adhesion. Alternatively, the accumulation of lymphocytes at the site of inflammation may have an effect on cell infiltration from the inside of blood vessels to the outside of blood vessels by the interaction of lymphocytes with vascular endothelial cells via adhesion molecules. Therefore
Compounds that inhibit the binding of # 414 to lymphocytes may be anti-inflammatory agents. Some metastatic cancers may have metastasized by binding to and invading the vascular endothelium,
It may be a cancer metastasis inhibitor. In addition, since # 414 is expressed in synovial nurse cells accumulating at the site of rheumatic lesions, this type of inhibitor may be an antirheumatic drug. In addition, macrophages and the like accumulate in the atherosclerotic lesions due to the binding between the vascular endothelium and the lymphocytes, which may be involved in the infiltration of these cells.

The present invention includes proteins that are functionally equivalent to human scavenger receptor-like protein # 414 (SEQ ID NO: 2). In the present invention, functionally equivalent proteins are called homologs. The homologue is specifically composed of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2; Protein consisting of an amino acid sequence). The number and location of mutations in the amino acid sequence
There is no limitation as long as the function of the protein can be maintained. The number of mutations is usually within 40% of all amino acids, preferably within 30% of all amino acids, more preferably within 20% of all amino acids.

The homolog of the present invention can be obtained by introducing a mutation into a DNA consisting of the nucleotide sequence encoding the amino acid sequence. In order to introduce a mutation into DNA, for example, `` Molecular Cloning: A Laboratory Manu
al Second Edition, Volume 1-3 "(Sambrook, J. et al. Cold Spring
Methods such as site-directed mutagenesis and PCR described in Harber Laboratory Press New York 1989) can be used. Using a suitable vector and host system, the DNA having the mutation introduced therein can be prepared, for example, using the method described in “Molecular Cloning: A
Laboratory Manual, 2nd edition, vol. 1-3, "may be used for genetic engineering.

The homolog in the present invention includes SEQ ID NO:
Encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence according to 1,
Proteins functionally equivalent to human # 414 (protein consisting of the amino acid sequence of SEQ ID NO: 2) are included. The DNA consisting of the nucleotide sequence of SEQ ID NO: 1 contains the nucleotide sequence encoding the amino acid sequence of scavenger receptor-like protein # 414 of the present invention. DNA that can hybridize under stringent conditions is likely to encode a structurally homologous protein. Accordingly, such proteins include many functionally equivalent proteins. In the present invention,
High homology means at least 60% or more, preferably 7% or more.
It refers to 0% or more, more preferably 80% or more sequence identity. Amino acid sequence homology can be determined using the BLAST search algorithm.

In the present invention, the stringent condition can be, for example, the following condition.
For example, when performing hybridization with a long probe (50 bases or more), the temperature (T
The standard of m) is obtained from the following formula, and the hybridization temperature can be set as follows. Tm = 81.5 + 16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (% G + C) -0.63 (% formam
ide)-(600 / n) -1.5 (% mismatch) [Na ⁺ ] is Na ion concentration [M], and n is the number of bases of the probe. Hybridization temperature = Tm−25 ° C. If a probe having 100% homology and 100 bases or more is used, the temperature conditions can be set as follows. (1) 65 to 75 ° C (without formamide added) (2) 35 to 45 ° C (in the presence of formamide) When using a short oligonucleotide as a probe, the following formula can be set as a guide. Hybridization temperature = Tm−5 ° C. = 2 ° C. × (number of A + T) + 4 ° C. × (number of C + G) −5 ° C.
After hybridizing under the above conditions, 0.2 × SSC, 0.5% SD
This shows that a positive signal is still observed even under the condition of washing at 65 ° C. in the S solution. The combination of these conditions is only an example. It is obvious to those skilled in the art that various factors that determine the stringency of hybridization are adjusted and the target stringency is empirically set. Factors that determine stringency include, for example, probe concentration and hybridization reaction time in addition to the above conditions.

The homolog of the present invention can also be obtained by using a gene amplification technique such as PCR. That is, a primer is designed based on the DNA sequence consisting of the nucleotide sequence of SEQ ID NO: 1, and encodes the protein of the present invention.
It is also possible to isolate a DNA fragment with high homology to the DNA sequence or a part thereof.

In the present invention, "functionally equivalent to human scavenger receptor-like protein # 414 (SEQ ID NO: 2)" indicates that the protein of interest has scavenger receptor activity. “Scavenger receptor activity” means the binding activity to a ligand. Binding activity includes activities that mediate the uptake of ligand into cells.

For example, the sugar chain galactose / N-acetylgalactosamine (Gal / GalNA
The binding activity to c) is one of the important activities as a scavenger receptor-like protein. The protein of the present invention
In addition to binding to Gal / GalNAc, when expressed on the cell surface, it exhibits Gal / GalNAc uptake activity by endocytosis. Therefore, as a protein functionally equivalent to the protein of the present invention, a protein having Gal / GalNAc binding activity, or preferably a protein having Gal / GalNAc uptake into cells by endocytosis can be shown. .

These activities can be confirmed by a known method. For example, the binding activity to the protein of the present invention can be evaluated by a ligand blot or an immunoprecipitation method using an antibody against the ligand. In addition, in order to confirm the incorporation activity of the ligand by the protein of the present invention, the ligand is brought into contact with cells expressing the target protein on the cell membrane. If the ligand is taken up into cells via the target protein,
The protein is a scavenger receptor-like protein
Judged to be functionally equivalent to # 414.

The ligand according to the present invention can be obtained by the method for screening a ligand according to the present invention as described below. That is, a ligand for a scavenger receptor-like protein can be selected by observing the uptake activity of a candidate ligand compound by endocytosis in cells expressing the scavenger receptor-like protein. Methods for confirming the uptake of a ligand by endocytosis via a scavenger receptor-like protein are known. Such an analysis method will be specifically described later as a method for screening a ligand for a scavenger receptor-like protein according to the present invention.

Alternatively, in the present invention, “functionally equivalent to human scavenger receptor-like protein # 414 (SEQ ID NO: 2)” means that the target protein has not only scavenger receptor activity but also cell adhesion activity. means. Cell adhesion activity in the present invention,
It can be known by culturing cells expressing the target protein on the cell surface in a plastic container and observing the adhesion of the cells to the inner wall of the container. The cell adhesion activity provided by the scavenger receptor-like protein of the present invention shows resistance even when cultured in the presence of 15 mg / L cyclosporin A. Since integrin-mediated cell adhesion is markedly inhibited under the same conditions, it can be said that the cell adhesion activity of the scavenger receptor-like protein of the present invention is due to a different mechanism from integrin.

The scavenger receptor-like protein of the present invention can be used not only for biomaterials such as cells and tissues expressing # 414, but also for a recombinant obtained by incorporating a gene encoding the same into an appropriate expression system. Can also be obtained as In order to obtain a scavenger receptor-like protein by a genetic engineering technique, a gene encoding a scavenger receptor-like protein may be incorporated into an appropriate expression system and expressed as described later. On the other hand, a gene encoding a scavenger receptor-like protein can be obtained by amplifying by PCR or the like using a cDNA library of human nurse-like cells or vascular endothelial cells as a template.

The present invention relates to a DNA encoding the protein of the present invention or a homolog thereof. D of the present invention
NA is cDNA, genomic DN if it encodes the amino acid sequence of SEQ ID NO: 2 or a homolog thereof.
The origin, such as A or synthetic DNA, is not limited. For example, the DNA of the present invention can be obtained by chemically synthesizing a DNA comprising the coding region of the nucleotide sequence shown in SEQ ID NO: 1. Further, the DNA of the present invention can also be amplified by PCR using a cDNA library of a nurse-like cell or other cells / tissues as a template and primers designed based on the nucleotide sequence.

The homologue of the DNA included in the present invention is a DNA containing a nucleotide sequence having at least 80% or more, more preferably 90% or more homology with the coding region of the nucleotide sequence of SEQ ID NO: 1. is there. The homology of the base sequences that make up the DNA is determined by the gene analysis software GENETYX-SV / RC Ve
r4.01 (manufactured by Software Development Corporation) and the gene analysis program BLAST (Internet address http: //www.ncbi.n
lm.gov/BLAST: References Altschul, SF et al., J. Mo
l. Biol., 215, 403-410, 1990; Altschul, SF et al.,
Nucl. Acid. Res., 25, 3389-3402, 1997). Furthermore, by using a cDNA derived from another species as a template, it encodes a homolog according to the present invention.
DNA can also be amplified.

Alternatively, screening a cDNA library prepared from a nurse-like cell including a synovial nurse cell, or a cell expressing the gene of the present invention such as a vascular endothelial cell, using a DNA comprising the above nucleotide sequence as a probe. Thus, the DNA of the present invention can be isolated. P
When cDNA from another species is used in the same manner as CR, DNA encoding a homolog of human # 414 derived from another species can be isolated. Probes are hybridized under stringent conditions as described above. Non-human animals include, for example, mice and rats.

In addition, generally, eukaryotic genes include polymorphism (p.
olymorphism) (eg, Nishi,
T. et al. (1985) J. Biochem., 97, 153-159). One or more amino acids may be substituted by the polymorphism, or the amino acid may not change at all even though the nucleotide sequence changes. DNAs having mutations in the nucleotide sequence based on these polymorphisms are also included in the DNA of the present invention.

Using the DNA of the present invention, the protein of the present invention can be obtained as a recombinant. The operations required to produce a recombinant are described, for example, in "Molecula" described above.
It is described in detail in many references, such as r Cloning. Specifically, a translation initiation codon is added upstream of the DNA to be expressed, and a translation stop codon is added downstream. In addition, promoter sequences that regulate transcription (eg, tr
A control gene such as p, lac, T7, or SV40 early promoter can be added, and the plasmid can be inserted into an appropriate vector, replicated in a host cell, and produced a functional expression plasmid.
Suitable expression vectors include, for example, for bacteria
pY for yeast, such as pRSET, pET, pGEMEX, pKK233-2
Examples include ES2, pVL1393 and pFastBac1 for insect cells, and pEF-BOS, pSRα, pDR2, and pCR3.1 for animal cells.

The DNA of the present invention is useful for gene therapy. The protein encoded by the DNA of the present invention functions as a scavenger receptor-like protein. Therefore, by introducing the DNA of the present invention into a living body and expressing it, it is possible to compensate for, for example, a decrease in the processing function of a denatured product based on endocytosis. More specifically, the DNA of the present invention can be used for various aging-related aging-related diseases such as arteriosclerosis, diabetic vascular disorder, cataract, and Alzheimer's disease, which are caused by a decrease in the function of treating foreign matter and waste products in vivo. It can be used to treat diseases. The DNA of the present invention can be introduced into a living body by a known method. Techniques for introducing a foreign gene into a living body for the purpose of treatment are known.
For example, a vector for introducing the DNA of the present invention can be obtained by incorporating the DNA of the present invention into a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, or the like. The vector of the present invention can be introduced into a living body ex vivo, for example, by infecting the isolated lymphocytes or the like with this vector and returning it to the living body again.

Next, the expression plasmid is introduced into a suitable host cell to obtain a transformant. Examples of the host cell include prokaryotes such as Escherichia coli, unicellular eukaryotes such as yeast, and cells of multicellular organisms such as insects and mammals.
Preferably, it is a mammalian cell. Examples of the mammalian cell include a CHO cell, a 293 cell, and a COS7 cell.
By culturing this transformant, a protein can be produced.

The obtained protein was subjected to ammonium sulfate precipitation, affinity chromatography, ion exchange chromatography, gel filtration chromatography, which is well known to those skilled in the art.
Purification can be performed by reverse phase chromatography or hydrophobic chromatography alone or in combination (Imai e
etal. J. Biol. Chem., 271, 21514-21521, 1996). Alternatively, a purification method using affinity chromatography can be applied by expressing the protein of the present invention as a fusion protein with a protein having a binding affinity such as a His tag or HA tag.

The present invention relates to a DNA which hybridizes with the DNA of the present invention or its complementary strand,
It relates to DNA having a length of 4 nucleotides. Such a DNA can be used as a primer for synthesizing the DNA of the present invention, or as a probe for detecting the DNA of the present invention or mRNA transcribed thereof. Designing a nucleotide sequence of a primer based on a given nucleotide sequence can be usually performed by those skilled in the art. It is also obvious to those skilled in the art that an appropriate probe is designed by comparing a base sequence to be detected with a known base sequence. When the DNA of the present invention is used as a probe, it can be labeled by a known method using an affinity substance such as digoxigenin or biotin, or a fluorescent substance such as FITC or rhodamine. DNA with required base sequence
Can be obtained chemically or enzymatically.

The probe or primer according to the present invention can be used for detecting the DNA of the present invention or the mRNA to which it has been transcribed. mRNA is analyzed by Northern blot or RT
-It can be detected by the PCR method. Since scavenger receptor-like protein is strongly expressed in, for example, synovial tissue of RA patients, grasping the expression state in the cells is considered to provide important information for grasping the state of RA joint inflammation. . Alternatively, when the receptor is deficient, nurse cells may not normally mature and differentiate lymphocytes. Therefore, by using a part of the DNA of the present invention as a primer,
It becomes possible to detect an immune abnormality caused by the deficiency of the present receptor.

Using the information of the scavenger receptor-like protein gene determined by the present invention, a promoter and an enhancer can be isolated. The nucleotide sequence of the scavenger receptor-like protein gene of the present invention is as shown in SEQ ID NO: 1. As further confirmed in the examples, this gene is Chr.18p11.23
Mapped to -18p11.31. Therefore, genomic DNA of the scavenger receptor-like protein of the present invention can be isolated by screening a genomic library containing this region with a probe based on the nucleotide sequence of SEQ ID NO: 1. Based on the isolated genomic DNA, an expression control region such as a promoter or an enhancer can be obtained. The expression control region thus obtained can be used to isolate transcriptional regulatory factors in a living organism,
It can be used for the development of pharmaceuticals aimed at controlling expression.

Further, based on the nucleotide sequence of the novel gene revealed by the present invention, an antisense DNA capable of controlling the expression of the novel gene product is provided. The antisense DNA according to the present invention is useful for controlling a disease state caused by enhanced expression of the novel gene. Alternatively, it is an important tool for clarifying the function of the novel gene. To effectively inhibit target gene expression using an antisense sequence, the length of the antisense DNA should be at least 10
It is at least a base, preferably at least 15 bases.

For example, in RA, if the scavenger receptor-like protein supports abnormal growth of synovial nurse cells, the role of antisense of the scavenger receptor-like protein plays a large role in the treatment of RA. From the viewpoint of controlling gene expression, not only antisense but also ribozyme can be designed. That is, a ribozyme that recognizes RNA transcribed from the coding region of the DNA represented by SEQ ID NO: 1 and cleaves it can be designed. Antisense and ribozymes are useful for treating and analyzing pathological conditions of autoimmune diseases including RA.

The present invention further relates to an antibody against the protein of the present invention. The antibodies of the present invention include both monoclonal antibodies and polyclonal antibodies. These antibodies can be prepared based on a known method using the protein of the present invention or a fragment thereof as an immunogen. The monoclonal antibody of the present invention can be obtained, for example, as follows. Specifically, mice, rats, hamsters and the like are immunized using the protein of the present invention as an antigen. Lymphocytes are removed from the spleen or lymph nodes of the immunized animal, fused with myeloma cells, and
Stein's method (Nature, 256, 495-497, 1975), or a modified method of Ueda et al. (Proc. Natl. Acad. Sci.
USA, 79, 4386-4390, 1982) and the like. Among the obtained hybridomas, those that produce an antibody having the desired reaction characteristics are cloned, and then cultured to produce a monoclonal antibody. By selecting a monoclonal antibody that recognizes a structure characteristically found in a protein of an immunogen, a monoclonal antibody that specifically recognizes the protein can be obtained. The characteristic structure of the protein of the present invention can be inferred by, for example, selecting an amino acid sequence specifically found in the amino acid sequence constituting the protein of the present invention.

The monoclonal antibody according to the present invention can be made into a chimeric antibody by recombining the constant region into a human immunoglobulin molecule. A chimeric antibody is one of the monoclonal antibodies produced by genetic engineering. For example, a mouse / human chimera whose variable region (V) is a variable region derived from mouse immunoglobulin (VH, VL) and whose constant region (C) is a constant region derived from human immunoglobulin (CH, CL) Monoclonal antibodies are known. The constant region derived from human immunoglobulin can be used as a constant region of human immunoglobulin belonging to any of IgG, IgM, IgA and IgE classes. Preferably, a constant region of human IgG is used.

Further, by humanizing the framework in the variable region, a so-called humanized antibody can be obtained. Human antibodies (CDR-grafted antibodies) are also one of the monoclonal antibodies produced by genetic engineering. For example, the hypervariable region (Hypervar
An iable region (HV) is derived from a mouse, and a humanized antibody in which the framework region of the variable region (V) is derived from human immunoglobulin is known. The HV is also called a complementarity determining region (CDR), and is present at three places in the variable region. HV1 (CDR
1), HV2 (CDR2) and HV3 (CDR3). These regions are important regions that determine antigen binding specificity in the variable region. On the other hand, framework re
gion) is a region where the amino acid sequence diversity is relatively small in the V region. 4 with HV in the V region
Occupies one area. They are called FR1, FR2, FR3, FR4 respectively. A humanized antibody can also consist of only the variable regions. Alternatively, a complete immunoglobulin molecule can be formed by giving a structure derived from human immunoglobulin as the constant region.

In addition, a human antibody gene is introduced into a mouse by a transgenic technique for manipulating an animal embryo,
A method for producing human antibodies by immunizing the mouse has also been established (Lonberg N., et al., Nature 368:
856-859, 1994; Green, LLetal., Nature Genet. 7: 13-2
1,1994; Mendez, MJ, et al., Nature Genet. 15: 147-15
6,1997; Nikkei Science 1995, June issue, P40-50). The immunoglobulin molecules obtainable by this method are fully human immunoglobulin molecules. As described above, humanized immunoglobulins are partially or entirely included in the present invention, as long as the antibodies recognize the scavenger receptor-like protein of the present invention.

A chimeric antibody can be obtained, for example, in the following manner (Experimental Medicine (Extra Extra Number), Vol. 1.6, No. 1).
No. 0, 1988; Japanese Patent Publication No. 3-73280). First, an active VH gene (reconstructed VDJ gene encoding the H chain variable region) and an active VL gene (reconstructed encoding the L chain variable region) were obtained from the hybridoma producing the mouse monoclonal antibody of the present invention. Isolated VJ gene). On the other hand, a CH gene (C gene encoding an H chain constant region) and a CL gene (C gene encoding an L chain constant region) are obtained from DNA encoding human immunoglobulin. Next, the CH is arranged downstream of the VH so that it can be expressed and incorporated into an expression vector (such as pSV2neo). Similarly, for VL,
The CL is sequenced downstream so that it can be expressed, and is incorporated into an expression vector. VH + CL and VL + CL can be integrated on one vector or inserted into another vector. The expression vector thus prepared is introduced into a suitable host cell. When VH + CL and VL + CL are inserted into different vectors, cotransfect with the two vectors. To facilitate screening, a cell line that does not itself produce antibodies is advantageous as a host cell. Specifically, P3X63Ag8.653 cells (ATCC CRL1580) and
Myeloma cells such as SP2 / 0-Ag14 cells (ATCC CRL1581) can be used. The vector uses the DEAE-dextran method,
Transformation can be performed by the calcium phosphate method or the electroporation method. Transformed cells are first screened using the drug resistance gene inserted into the expression vector as an indicator. Further, a transformant expressing the desired antibody molecule can be screened to obtain a desired chimeric monoclonal antibody-producing cell.

Furthermore, a humanized antibody can be obtained, for example, as follows (Japanese Patent Application Laid-Open No. 4-506458 and Japanese Patent Application Laid-Open No. 62-296890). First, the mouse H chain CDR gene and the corresponding mouse L chain CDR gene are isolated. On the other hand, from the human immunoglobulin gene, a human H chain gene encoding all regions other than the human H chain CDR corresponding to the mouse H chain CDR, and a human L chain gene corresponding to the mouse L chain CDR are isolated. The isolated mouse H chain CDR gene and human H chain gene are introduced into an appropriate vector so that they can be expressed. Similarly, a mouse L chain CDR gene and a human L chain gene are introduced into another suitable expression vector so that they can be expressed. Or
The mouse H chain CDR / human H chain gene and the mouse L chain CDR / human L chain gene can be introduced into the same expression vector so that they can be expressed. By transforming an appropriate host cell with the expression vector thus prepared, a humanized antibody-producing transformed cell can be obtained. Transformation and screening of host cells can be performed similarly to chimeric antibodies.

A polyclonal antibody is described, for example, in “An
tibodies; A Laboratory Manual, Lane, HD et al.
ld Spring Harber Laboratory Press New York 198
9 years).
In other words, by immunizing an animal with an appropriate method using the purified protein of the present invention or a fragment thereof as an antigen, an antibody that specifically recognizes the antigen protein can be easily prepared and purified. it can. As an immunized animal for obtaining a polyclonal antibody, a mouse, rat, hamster, rabbit, or the like is generally used. In any case, in order to use the protein fragment for the immunogen, the immunogenicity can be enhanced by binding to a carrier protein such as KLH or albumin.

The antibody of the present invention can be used for detecting the protein of the present invention. A method for detecting a protein in a sample using an antibody is known as an immunoblot method, an immunoassay method, an immunostaining method, or the like. In addition, a method of directly or indirectly labeling an antibody for use in these detection methods is also known. Signs include:
Generally, enzymes, radioisotopes, or fluorescent substances are used. The protein of the present invention is considered to be expressed in nurse-like cells involved in diseases such as RA. Therefore, its immunological detection provides important information in the diagnosis of these diseases. The antibody of the present invention can be used as a reagent kit for detecting the scavenger receptor-like protein of the present invention by appropriately combining a solvent and a solute such as a buffer, a stabilizer, a suspending agent, and a salt.

The present invention further relates to a method for detecting the binding activity of a test compound to the protein of the present invention, and a method for screening a test compound having an activity for binding to the protein of the present invention based on the detection method. The detection method of the present invention includes the following steps. a) a step of bringing a test compound into contact with the protein of the present invention; b) a step of detecting the binding between the protein and the test compound; and selecting the test compound that binds to the protein based on this method, thereby obtaining the protein of the present invention. A test compound having an activity of binding to is screened.

The proteins used for screening include:
Not only natural proteins but also recombinant proteins can be used. On the other hand, the test compound may be, but is not limited to, a library of low-molecular organic compounds or proteins, or a component derived from animals, plants and microorganisms. The binding between the protein of the present invention and a test compound can be easily detected by immobilizing either one on a solid phase and labeling the other (in vitro).
Therefore, for example, a combinatorial library synthesized on a support is a test compound suitable for the screening of the present invention. In this case, by reacting the fluorescently labeled protein of the present invention, the binding between the two can be easily detected.

According to the screening of the present invention, for example,
It is possible to isolate the ligand of the scavenger receptor-like protein of the present invention. The scavenger receptor-like protein of the present invention is considered to have a function of taking up a ligand into cells in a living body. Therefore,
The protein of the present invention can be identified by the screening of the present invention. Further, by applying this screening, it is also possible to obtain agonists and antagonists of the scavenger receptor-like protein of the present invention.

Further, the activity as a ligand can be detected using the incorporation of a candidate ligand compound into cells via the scavenger receptor-like protein of the present invention as an index. Further, based on this detection method, a ligand can be screened from the test compound.
That is, the present invention relates to a method for detecting the activity as a ligand for the scavenger receptor-like protein of the present invention, and a method for screening a ligand based on this method. In the present invention, the activity as a ligand means an activity that binds to the protein of the present invention expressed on the cell surface and is taken up into the cell. The method for detecting the activity as a ligand according to the present invention specifically includes the following steps. a) a step of bringing a candidate compound for a ligand into contact with cells expressing the protein of the present invention; b) a step of detecting uptake of the candidate compound by endocytosis via the protein; and uptake by endocytosis via the protein. By selecting a compound to be used, a compound that acts as a ligand for the protein of the present invention can be screened.

Incorporation of a candidate compound into a cell by endocytosis is performed by tracking the intracellular localization of a scavenger receptor-like protein and a ligand candidate compound. Specifically, for example, by expressing a scavenger receptor-like protein as a fusion protein with a labeled protein such as GFP, the intracellular localization can be clarified. On the other hand, for ligands, FITC and Dioctadecyl-tetramethyl-indocarbocyani
It can be tracked by labeling with n (DIL) or biotin. The observation that the scavenger receptor-like protein moves from the cell membrane to the inside of the cell together with the ligand is evidence that the candidate ligand compound has been taken up into the cell by endocytosis. Using such an analysis method, a ligand for the scavenger receptor-like protein of the present invention can be screened.

In the ligand screening method of the present invention, the following compounds can be used as ligand candidate compounds. The scavenger receptor generally uses, as a ligand, a protein that undergoes a structural change due to oxidation, saccharification, or the like. Such proteins include, for example, proteins directly oxidized by various enzymes (such as myeloperoxidase and lipoxygenase) in the field of inflammation, and proteins oxidized and denatured by active oxygen secondary produced by the action of these enzymes, or cancer. And denatured (heterologous) proteins caused by aging and aging, and glycation products (Advanced glycati
on End Product: AGE). In particular,
For example, serum albumin modified by maleylation or glycation (Glycated) can be shown. Furthermore, MDA-LDL (malondialdehyse-treated LDL) and the like can be indicated as denatured proteins. These compounds are used as candidate compounds for the ligand screening method of the present invention. Since the scavenger receptor-like protein according to the present invention has a C-type lectin that binds to a sugar chain and has a structurally characteristic sequence for galactose recognition, glycoproteins and glycolipids are also candidate ligands. It is thought that it can be raised. Such compounds include so-called proteoglycans, which are covalently bonded compounds of glucosaminoglycans (mucopolysaccharides) such as keratan sulfate and heparan sulfate with proteins.

The present invention further relates to a method for detecting the activity of a test compound to inhibit the binding between the scavenger receptor-like protein of the present invention and its ligand, and a method for screening a compound that inhibits the binding to a ligand based on this method. The detection method of the present invention includes the following steps. a) a step of bringing the protein of the present invention into contact with the ligand in the presence of the test compound, or a ') a step of bringing the protein of the present invention into contact with the test compound in advance and then contacting with the ligand; Step of Detecting Binding to Ligand By selecting a compound that inhibits the binding, a compound that inhibits the binding between the scavenger receptor-like protein of the present invention and its ligand can be screened.

[0078] The scavenger receptor-like protein, ligand, and
Alternatively, test compounds similar to those described above can be used. Screening in vivo or in
The same can be done in vitro. Ie in vivo
The screening method in includes the following steps. a) a step of bringing the modified protein into contact with a cell expressing the scavenger receptor-like protein of the present invention in the presence of the test compound; A step of subsequently contacting the modified protein, b) a step of detecting the uptake of the modified protein into cells, and c) a step of selecting a compound that inhibits the uptake.

[0079] Incorporation of the modified protein into cells can be detected by labeling the modified protein. For example, a method of incubating cells expressing the scavenger receptor-like protein of the present invention with a radiolabeled modified protein and a test compound and measuring the change in radioactivity incorporated into the cells can be used. By the above-described method for screening a substance that inhibits binding, screening of agonists and antagonists for binding between the scavenger receptor-like protein of the present invention and its ligand is performed.

In the case of the exclusion-type binding experiment, a fixed amount of a labeled ligand and various concentrations of unlabeled agonist and antagonist candidate compounds are reacted with a fixed amount of cells expressing the present receptor. After washing, the radioactivity, enzyme activity, or fluorescence intensity of the cells is measured. Thereby, the binding inhibitory activity of the agonist and the antagonist can be examined. In the case of a saturation type binding experiment, various concentrations of a labeled ligand and a predetermined amount of unlabeled candidate agonist and antagonist compounds are reacted with cells expressing a certain amount of the present receptor. After washing, the radioactivity, enzyme activity, or fluorescence intensity of the cells is measured. Thereby, the binding inhibitory activity of the agonist and the antagonist can be examined.

The compound selected by the screening method of the present invention is useful as an agent for controlling the activity of the scavenger receptor-like protein of the present invention. When the protein of the present invention is involved in cell adhesion, cell adhesion can be inhibited by a ligand for this protein or a compound that inhibits the binding between this protein and its ligand. Therefore, by using these compounds as a medicine, it becomes possible to prevent cancer metastasis. The drug of the present invention can be produced by mixing the compound selected by the screening method as an active ingredient and mixing it with a physiologically acceptable carrier, excipient, diluent, or the like. The agent of the present invention
For the purpose of preventing or treating the above various diseases involving the scavenger receptor-like protein of the present invention, it can be administered orally or parenterally. Oral preparations can be in the form of granules, powders, tablets, capsules, solvents, emulsions, suspensions and the like. As parenteral preparations, dosage forms such as injections, drops, external preparations, suppositories and the like can be selected. Injections include subcutaneous injections, intramuscular injections, intraperitoneal injections, and the like.

The dose is appropriately selected according to the method of administration (formulation) and the condition of the administration subject (physique, age, sex, symptoms). In general, the effect can be obtained by adjusting the dose to 1 μg to 100 mg, more preferably 1 μg to 50 mg, per oral administration per kg of body weight. The number of administrations can be appropriately selected, for example, in the range of 1 to 3 times a day.
However, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, or a dose exceeding the above range may be necessary. If the compound can be encoded by DNA, the DNA may be incorporated into a vector for gene therapy to perform gene therapy.

The present invention also provides a transgenic non-human vertebrate in which the expression of the scavenger receptor-like protein of the present invention has been modified or is capable of inducing the modification. In the present invention, “alteration of expression” includes enhancement and attenuation of expression. In addition, expression (ectopic expression) in tissues and cells different from the original is included. Also, a transgenic non-human vertebrate (knockout animal) modified so that the homolog of the scavenger receptor-like protein of the present invention in the model animal is not expressed at all is included. The expression of these proteins may be derived from cDNA, genomic DNA, or synthetic DNA. Further, "alteration of protein expression" includes alteration of both transcription and translation steps. Therefore, the transgenic animal of the present invention includes an animal in which the scavenger receptor-like protein gene of the present invention is artificially introduced into germ cells by gene recombination, an antisense RNA.
Antisense transgenic animals in which the function of a specific gene has been suppressed using, an animal in which the scavenger receptor-like protein gene of the present invention has been knocked out, an animal in which a point mutation DNA has been introduced into the scavenger receptor-like protein gene of the present invention, and the like. included.

The present invention also relates to the case where the alteration of the expression is site-specific, the case where the alteration is specific to a specific stage of development or life cycle, and the case where the alteration is induced in response to an artificial stimulus. included. Specifically, by introducing the gene of the present invention or its antisense under the control of a tissue- or cell-type-specific promoter, or a time-specific promoter, a site-specific expression modification is achieved. It is possible. On the other hand, it is also possible to induce expression modification by artificial stimulation using a promoter which is responsive to an external stimulus such as a drug.

For example, by preparing a transgenic animal in which an expression vector encoding the protein of the present invention has been integrated into a chromosome, the expression of these proteins can be increased, and the expression pattern and distribution can be altered. . In addition, by introducing mutations into the expression control regions of these endogenous genes or by adding or substituting other expression control regions, the transcription level is artificially increased as compared to the expression level of the original gene, Downward or alteration of expression pattern or distribution can be performed. On the other hand, translation into a protein can also be modified by deleting a part of an exon or replacing it with a termination codon by introducing a point mutation into the translation region. Also antisense
By expressing RNA or ribozyme, it is also possible to control the expression of the gene encoding the protein of the present invention. These mutations can be introduced by a known method. A general method for obtaining a knockout mouse or a transgenic mouse using a mouse as a model animal is described below.

For example, a knockout mouse in which an endogenous mouse gene corresponding to the human # 414 gene has been inactivated can be prepared as follows. (1) Construction of targeting vector Human # 414 cDNA cloned according to the examples is labeled with ³² P, and a hybridization probe is prepared according to a standard method. Using this probe, a phage library (Clontech) into which mouse genomic DNA has been introduced is screened, and a mouse # 414 genomic DNA clone containing an exon encoding mouse # 414 protein is obtained. Next, the plasmid pBluescriptIISK (-) was added to the diphtheria toxin gene (as a negative selection marker; Yagi
T, et al., Proc. Natl. Acad. Sci., Vol. 87, 9918-9.
922, 1990) and mouse # 414 genomic DNA. This includes the neomycin resistance gene ("Neo" as a positive selection marker; McBurney MW, et a
l., Nuc. Acids Res. Vol. 19, 5755-5761, 1991)
It is inserted and ligated into exons in the mouse # 414 genomic DNA. The obtained plasmid is cut with NotI to linearize it to obtain a targeting vector.

(2) Introduction of targeting vector into ES cells Mouse embryonic stem cells (3 × 10 ⁷ cells) cultured in a DMEM medium containing 15% fetal bovine serum
Is treated with trypsin to form single cells, and washed three times with a phosphate buffer to adjust the cell concentration to 4 × 10 ⁷ cells / ml. 40 μg of the targeting vector is added per 1 ml of the cell suspension, and an electric pulse is applied once under the condition of 200 V / cm (3 μF). Then, 5 × 10 ⁶ ES cells were seeded on a Petri dish (10 cm), cultured in a maintenance medium for 1 day, and then the medium was changed to a selection medium (G4
18: 125 μg / ml). Thereafter, the medium is replaced every day to obtain a neomycin-resistant ES cell clone. By separately culturing the obtained ES clones in a 24-well plate on which Feeder cells are spread, a replica of neomycin-resistant ES cells can be obtained.

(3) Screening of knockout ES cells For each of the obtained neomycin-resistant ES cells, it was determined whether or not disruption (knockout) of the endogenous gene encoding mouse # 414 protein by homologous recombination had occurred. Confirm by blotting. ES obtained by knocking out the endogenous gene thus obtained
Cell clones are used to generate knockout mice.

(4) Preparation of Knockout Mouse Embryos obtained by crossing male and female C57BL / 6J (purchased from CLEA Japan) were obtained from each of the ES cell clones obtained by knocking out the endogenous mouse # 414 gene obtained above. 15 per embryo per blastocyst
Microinjection (Proc.Natl.Acad.Sci.
USA, Vol 77, 7380-7384, 1980; Hogan B, Costantini F and
Lacy E, Manipulating the mouse embryo: A laborato
ry manual, Cold spring harbor laboratory press, 19
86) Do it. Immediately after injection, foster parent IC 2.5 days after pseudopregnancy
Approximately 10 blastocysts per side of the uterus are implanted into the uterus of an R mouse (purchased from CLEA Japan). Thus, each chimeric mouse is obtained. Next, each of the obtained chimeric mice is bred with normal C57BL / 6J mice to obtain an agouti-colored mouse with a hair-color gene derived from ES cells. The genotype of agouti-colored mice is confirmed by genomic Southern blotting to obtain hetero-knockout mice. By crossing the hetero knockout mice, a homo knock out mouse can be obtained according to Mendel's law.

A conditional knockout mouse in which a mouse endogenous gene corresponding to the human # 414 gene has been site-specifically inactivated can be prepared as follows. (1) Construction of targeting vector Plasmid pBluescriptIISK (-) contains diphtheria toxin gene (as a negative selection marker; Yagi
T, et al., Proc. Natl. Acad. Sci., Vol. 87, 9918-99.
22, 1990) and mouse # 414 genomic DNA are ligated and inserted. The neomycin resistance gene sandwiched between two loxP genes ("Neo" as a positive selection marker; McBurney MW, et al., Nuc. Acids Re
s. Vol. 19, 5755-5761, 1991) is inserted and ligated into the intron in the mouse # 414 genomic DNA. Another loxP gene is inserted into another intron. loxP
The gene is cut out from plasmid pBS246 (Gibco BRL) and used. The obtained plasmid is cut with NotI to linearize it to obtain a targeting vector.

(2) Conditional knockout ES
Production of cells The obtained targeting vector was introduced into ES cells in the same manner as in the production of knockout mice, and mouse # 414 was introduced.
Within the intron of the endogenous gene encoding the protein,
A neomycin resistance gene sandwiched between two loxP genes is inserted, and one loxP is inserted into another intron.
ES cells into which the xP gene has been inserted are selected by genomic Southern blotting. Then, using the same method as the introduction of the targeting vector, Cr
e The Cre is transiently expressed by introducing the expression vector pBS185 (Gibco BRL) in a circular form. Fees obtained ES clone
Cloning is performed by separately culturing each in a 24-well plate on which der cells are spread. Next, ES cells in which the neomycin resistance gene sandwiched by loxP was excised by Cre expression and only two loxP genes were integrated into separate introns on the endogenous gene were analyzed by Southern hybridization and PCR. Sort out.

(3) Preparation of Conditional Knockout Mouse Using each of the ES cell clones obtained above, a heterozygous mutant mouse is obtained in the same manner as in the preparation of a knockout mouse. This mouse was then bred with a transgenic mouse that expresses Cre in a site-specific manner,
Mice having the # 414 gene heterozygously mutated and further having a Cre-expressing gene are selected by Southern hybridization. By mating this mouse with a heterozygous mutant mouse of the endogenous # 414 gene, a conditional knockout mouse can be obtained. This mouse has its own Cr
Cre is site-specifically expressed by the e-expressed gene, and the endogenous # 414 gene sandwiched by the loxP gene is knocked out only at that site.

Further, the transgenic mouse according to the present invention can be prepared as follows. (1) Construction of Insertion Gene The human # 414 cDNA cloned in Example and a promoter gene are inserted into a plasmid pBluescriptIISK (-). Using a restriction enzyme at the multicloning site of this plasmid, a linear gene inserted so that the human # 414 gene can be expressed downstream of the promoter gene is cut out.

(2) Preparation of Transgenic Mice Transgenic mice were prepared by microinjecting the above-mentioned gene into the pronucleus of a fertilized egg (one-cell egg) obtained from C57BL / 6J (Clea Japan) (Hogan B) , Co
stantini F and Lacy E, Manipulating the mouse embr
yo: A laboratory manual, Cold spring harbor labora
tory press, 1986). Immediately after the injection, about 10 blastocysts per side of the uterus are implanted into the uterus of a foster parent ICR mouse (purchased from CLEA Japan) 1.5 days after pseudopregnancy. Then, a founder mouse (child mouse) is born from the foster parent mouse. From the founder mice, mice in which the target gene is integrated on the chromosome are selected by Southern hybridization. A transgenic mouse can be obtained by mating the selected mouse with a normal mouse.

The transgenic mouse (Tg) is a technique for analyzing a phenotypic abnormality due to overexpression (gain of function), and the knockout mouse (KO) is a technique for analyzing a loss of function. For example, a knockout mouse is created by destroying (inactivating) the mouse # 414 gene based on the analysis information of the mouse homolog of the # 414 gene. Next, the function of this gene can be elucidated by analyzing the biological and pathological characteristics of the knockout mouse. Furthermore, by introducing the human # 414 gene into this knockout mouse, human-derived # 4
Transgenic mice expressing 14 genes can be created. By using the obtained transgenic mouse, the effect of a drug targeting the human-derived # 414 gene can be evaluated in vivo. For example, when this gene is involved in the development of arteriosclerosis or diabetic vascular disorders, the transgenic mouse can be used as a model animal for these diseases. Depending on the model animal, it is possible to evaluate the onset-suppressing effect and therapeutic effect of agonists and antagonists. Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

[0096]

EXAMPLES General experimental procedures used in each step of the present invention (DNA agarose gel electrophoresis, ligation reaction, transformation of E. coli, digestion of DNA with restriction enzymes,
Northern analysis etc.) are described in “Current Protocols in Molecular B
iology "(FM Ausubel et al. Ed., John Wiley & So
ns. Inc.).

Example 1 Isolation of Human # 414 cDNA (1) Nurse Cell-Specific Gene Isolation Human nurse cell line Hs67 (ATCC No HTB163), non-nurse cell line IMR-32 (ATCC No CCL127), SK- HEP-1 (ATCC No
HTB52), A549 (ATCC No CCL185), Hs913T (ATCC No
HTB152) cells from the AGPC method (Analytical Biochemistry)
(1987) 162, 156-159) and total RNA was prepared.
MRNA was purified using ATtract system 1000 (promega). Hs67, IMR-32, SK-HEP-1, A549, Hs913T cells
Not I to synthesize first strand cDNA from mRNA
primer-adapter (SuperS made by LIFE TECHNOLOGIES, INC.)
cript Plasmid System). 2 μg of mRNA of each cell
MMLV RTas after annealing Not I primer-adapter
e (SuperScript Plasmid manufactured by LIFE TECHNOLOGIES, INC.)
System) and the first s
A trand cDNA was synthesized, followed by a second strand cDNA. The synthesized second strand cDNA was extracted with phenol / chloroform, precipitated with ethanol, dried and dissolved in 5 μl of TE. Next, Hs67 cells, a nurse cell line (tester)
Expressed in non-nurse cell lines IMR32, SK-HEP2, A549, Hs91
To obtain mRNA not expressed in 3T cells (driver), tester cD using a commercially available subtraction kit
NA homogenization and subtraction with the driver cDNA were performed. This is further processed by the RDA method (Representational differ
A subtraction cDNA was prepared by re-subtraction by PCR and amplification of the subtraction cDNA by PCR (Suppression Subtractive Hybrid).
idization-RDA; SSH-RDA).

[0098] Specifically, CLONTECH PCR-Select ™ cDN
Kit Ins using A Subtraction Kit (CLONTECH)
Subtraction and PCR were performed according to the truction manual. The tester Hs67 cDNA synthesized from 2 μg of the above-described mRNA and the cDNA mixture derived from the above four cell lines as the driver were digested with RsaI, the adapter attached to Kit was added, and the first subtraction was performed using the primer attached to Kit. (1sthybrdization / 2nd hybridizatin) and 1st PCR were performed. For the obtained PCR product,
The RDA method was performed.

Next, the operation of the RDA method will be described below. First, the obtained PCR product was digested with an RsaI restriction enzyme to cut an adapter, and purified with a PCRprep purification kit (Promega). Next, a new adapter AD2 was ligated to the purified cDNA fragment. For ligation, 50ng /
Add 2 μl of purified cDNA and 2 μl of AD2 adapter (10 μM), 2 μl of buffer included in the kit, 3 μl of distilled water, and 1 μl of T4 DNA ligase included in the kit to make a total volume of 10 μl.
And left overnight. AD2 is AD2S (sequence 5'-CAGCTCCACAACCTA
CATCATTCCGT 26mer) (SEQ ID NO: 3) and AD2A (sequence 5'-
(ACGGAATGATGT 12mer) (SEQ ID NO: 4) obtained by annealing AD2 at 65 ° C. at a final concentration of 10 μM. AD2 added PCR fragment 300 pg and RsaI digested IMR-32, SK-HEP-1, A54 as driver
9. Mix the mixture cDNA fragment derived from Hs913T cells.
1st subtraction of cDNA fragment derived from each cell used for driver
2 μg of poly (A) RNA according to the kit manual in the same way as ion
(Mixed with 0.5 μg of each cell-derived mRNA) and converted into cDNA, and one fifth (about 0.5 μg) of RsaI-digested
Equivalent) was used.

CLONTECH PCR-Select ™ cDNA Subtractio
n Using the hybridization buffer provided with Kit (CLONTECH), make the total volume 4 μl, put mineral oil on, and hybridize at 68 ° C overnight (2nd subtra
ction). Using 0.2 μl of these, PCR was performed with 50 μl of the AD2S primer at a final concentration of 1 μM. For PCR, pfu polymerase (manufactured by STRATAGENE) was used. First, 72 ℃, 5
The ends of the annealed fragments were repaired. Further, denaturation was performed 22 times at 94 ° C for 1 minute, annealing at 60 ° C for 1 minute, and extension at 72 ° C for 3 minutes (2nd PCR). The obtained PCR product was further similarly digested with RsaI, purified, and ligated with an AD3 adapter. Ligation was performed using 2 µl of the purified PCR product (50 ng), 2 µl of AD3 adapter (10 µM), 2 µl of buffer included in the kit, 3 µl of distilled water, and T4 included in the kit.
1 μl of DNA ligase was added to make the total volume 10 μl, and the mixture was allowed to stand at 16 ° C. overnight.

AD3 is AD3S (sequence 5'-GTCCATCTTCTCTCTGAGAC
TCTGGT 26mer) (SEQ ID NO: 5) and AD3A (sequence 5'-ACCAG)
AGTCTCA 12mer) (SEQ ID NO: 6) was prepared by annealing an oligonucleotide. PCR with AD2
10pg fragment and 1st and 2nd subtraction as driver
RsaI digested IMR-32, SK-HEP-1, A549, Hs used for
Mix the 913T cell-derived cDNA fragment equivalent to 0.5 μg, and add 2nd subtra
The total volume was made 4 μl in the same manner as in ction, and mineral oil was placed on the plate, followed by hybridization at 68 ° C. overnight (3rd subtractio).
n). Using 0.2 μl of this, add 2 μl with AD3S primer.
PCR was performed under the same conditions as for nd PCR (3rd PCR). Got
After recovering the PCR product as 4 fragments of different sizes by 3% agarose electrophoresis, Taq polymerase (TaKaR
After treating in a) and adding A to the end, the clone was cloned into PCR3.1 and transformed into Escherichia coli DH5α. Among the obtained clones, arbitrarily insert the inserts of 300 clones by PCR.
And digested with restriction enzymes HaeIII and MspI, and several fragments having different restriction enzyme digestion patterns were obtained by 3% agarose electrophoresis. The nucleotide sequence of each fragment was determined, and based on that nucleotide sequence, it was considered a novel gene # 4
I chose 14.

(2) Identification of full-length human # 414 cDNA In order to obtain an isolated full-length human # 414 gene cDNA, a radiolabeled # 414 cDNA fragment was used as a probe for a human kidney-derived cDNA library (manufactured by CLONTECH). To perform colony hybridization. When the cDNA chain length of the four clones showing a positive signal was examined by digestion with the restriction enzyme EcoRI, one of the clones had a maximum length of about 2 kb, and the nucleotide sequence of this clone was determined by ABI PRISM 310 Geneti.
c analyzer (Applied Biosystems) and ABI PRI
SM dRhodamine Terminator CycleSequencing Ready Rea
ction Kit (Applied Biosystems). Since the clone was shorter than the mRNA size by the Northern analysis performed earlier, 5'-
We attempted to clone the upstream region of the gene by RACE.

First, the following primers required for 5'-RACE were synthesized based on this DNA sequence. 414R4 5'-AAGAAAAACCTGCTGCAGATTTTG-3 '(SEQ ID NO:
7) 414R5 5'-GTCGTTCTTGATTCGCTGGATAGC -3 '(SEQ ID NO:
8) The template for 5'-RACE is a single-stranded cDNA library derived from human lung M
arathon-Ready cDNA (manufactured by CLONTECH) was used. PCR
The reaction was performed using 5 μl of the above template and the primer AP1 (manufactured by CLONTECH).
(Final concentration 1 pmol / μl) and 414R4 (SEQ ID NO: 7)
(Final concentration 1 pmol/μl) and TaKaRa La Taq (5U)
The attached buffer of 2.5 mM dNTPs and TaKaRa was used (50 μl in total volume). The reaction is once at 94 ° C for 3 minutes, at 94 ° C for 1 minute, at 65 ° C for 1 minute.
30 minutes at 72 ° C. for 2 minutes and once at 72 ° C. for 3 minutes. Thereafter, the sample was subjected to 2% agarose electrophoresis, a fragment of about 0.6 kb was recovered from the gel, purified by PCR-PREP (manufactured by PROMEGA), and further purified by Sec.
Used as a template for ond PCR. Primer AP2 (CLONTE
CH) and 414R5 (SEQ ID NO: 8) using TaKaR
a La Taq (5U) and Buffer with 2.5mM dNTPs and TaKaRa
er (total volume 50 μl). The reaction is performed once at 94 ° C for 3 minutes,
Min, 65 ° C for 1 minute, 72 ° C for 2 minutes 20 times, and 72 ° C for 3 minutes once. The obtained PCR fragment was subjected to 2% agarose electrophoresis, and was
A 6 kb fragment was recovered from the gel, purified by PCR-PREP (manufactured by PROMEGA), and inserted into a PCR2.1 vector (manufactured by INVITROGEN). Ten clones were arbitrarily selected from the obtained clones, and their base sequences were sequenced using ABI PRISM 310 Genetic analyzer
d Biosystems) and ABI PRISM dRhodamine Term
inator Cycle Sequencing Ready Reaction Kit (Appli
ed Biosystems). Based on the determined nucleotide sequence, the nucleotide sequence shown in SEQ ID NO: 1 was finally determined as the nucleotide sequence of # 414.

[Example 2] Localization of # 414 gene on chromosome This nucleotide sequence was obtained from Gene
The position of the # 414 gene on the human chromosome was estimated using the Bridge4-Radiation Hybrid Panel (RH mapping). The # 414 gene-specific primer used was 414-F4
A (5'-CAG ATG GAGAAC ATC ACC-3 '/ SEQ ID NO: 11) 4
14-R7 (5'-CTT CAT TTC TTC CAT AAT CA-3 '/ SEQ ID NO:
Using 12), PCR was performed as follows. The PCR reaction composition was 200 ng for each primer and 50 ng for genomic DNA template.
Takara Shuzo Taq polymerase 1μl and 10X buffer 3μ
1 and 3 μl of dNTP were added to make the total volume 30 μl. The reaction temperature is
Perform 35 cycles of 1 minute each at 94 ° C, 55 ° C, and 72 ° C.
Minutes went. The chromosomal locus was mapped to Chr.18p11.23-18p11.31 according to the sample in which the band of the correct size was obtained and the pattern of the sample number in which the band was not obtained.

[Example 3] Analysis of deduced amino acid sequence of human # 414 The determined nucleotide sequence of human # 414 cDNA (SEQ ID NO: 1) and an open reading frame having no internal termination codon
The amino acid sequence of (ORF) (SEQ ID NO: 2) is shown in FIGS. It has been revealed that the human # 414 gene has an ORF consisting of 742 amino acid sequences, and has a highly hydrophobic amino acid (underlined) at one position. It is presumed that there is. SEQ ID NO: 1 shows the amino acid corresponding to the nucleotide sequence. Amino acid sequence similarity analysis was performed using gene analysis software GeneWorks (manufactured by Intelli Genetics). Human scavenger receptor SRA-I that has homology to human # 414 and is structurally similar
FIG. 3 shows the results. Conserved amino acids are indicated by *. Although the homology between human # 414 and the human class AI scavenger receptor (SRA-I) was 16% in total, human # 414 was similar in basic domain structure as shown below. Was predicted to be a member of the scavenger receptor family.

FIG. 4 shows the predicted structure of the human # 414 protein. Although human # 414 had low homology in amino acid sequence, its structural characteristics were completely identical to that of SRA-I (class AI scavenger receptor). That is,
First, there is a cytoplasmic region consisting of 37 amino acids in the N-terminal region, and a sequence (YXRF) having a hook structure considered to play an important function in endocytosis is present in that region.
Subsequently, a region having a high hydrophobicity, which may be a transmembrane region consisting of 22 amino acids, a spacer region consisting of 383 amino acids and a coiled coil-like region are found. In the coiled coil-like region, there was a leucine zipper structure in which a hydrophobic amino acid such as leucine appeared every 7 amino acids. In addition, there was a collagen-like region consisting of 147 amino acids, and 49 repetitions of the GXX sequence were found in this region. Furthermore, a large number of basic amino acids lysine or arginine considered to be important for binding to the ligand were present at the X position of GXX. Histidine, which is essential for dissociation of the ligand in the endosome, was also present. On the other hand, there is a type-specific region consisting of 153 amino acids at the C-terminal. In # 414, it was confirmed that this region was not a cysteine-rich domain found in conventional SRA-I, MARCO, etc., but a lectin-like domain involved in sugar chain binding. These results suggest that the scavenger receptor-like proteins of the present invention belong to the class A scavenger receptor family in which the C-terminal type-specific region has been replaced by a lectin-like domain.

Example 4 Expression Analysis of Human # 414 mRNA by Northern Blot Analysis To examine the expression of human # 414 mRNA, the following 14 types of human cell lines, human tissues, or human primary vascular endothelial cells ( Northern blot analysis was performed using Clonetics as a sample. Of the following cell lines, RA189SM is a human synovial cell line. Human cell line: Nurse cell RA189SM (Takeuchi E., et al, Arthritis Rhe
um.42: 221-228,1999) Nurse cell Hs67 (ATCC No HTB163) Fibroblast-like cell IMR32 (ATCC No CCL127) Promyeloma cell HL60 (ATCC No CCL240) T-cell leukemia cell Jurkat (ATCC No TIB152) ) B lymphoma cells MC / CAR (ATCC No CRL8083) Fibroblast-like cells Hs683 (ATCC No HTB138) Epithelial-like cells SIHA (ATCC No HTB35) Epithelial-like cells A549 (ATCC No CCL185) Epithelial-like cells SK-HEP-1 ( ATCC No HTB52) Fibroblast-like cells Hs913T (ATCC No HTB152) Fibroblast-like cells SK-LMS-1 (ATCC No HTB88) Nurse cells HSN27E (Iwagami S. et al, J. Immunol., 153:
2927,1994) Fibroblast-like cells FHs173WE (ATCC No HTB158) Fibroblast-like cells Hs729T (ATCC No HTB153) Human tissues: heart, brain, placenta, lung
(lung), liver (liver), skeletal muscle (skeletal muscle), kidney (kid)
ney), pancreas, spleen, thymus, prostate, testis, testis, ovary, small intestine (smal)
l intestine), colon (colon), and peripheral blood lymphocytes (PB
L) Human primary vascular endothelial cells: Umbilical vascular endothelial cells (HUVEC) Aortic endothelial cells (HAEC) Cardiac coronary endothelial cells (HCAEC) Total RNA from the above cells using TRIzol ^™ (LIFE TECHNOLOGIES, INC.) Prepared. 15 μg of the total RNA of the prepared human cell line was electrophoresed on a 1% agarose gel containing formaldehyde, and the nylon membrane Hybond ^TM -N +
(Amersham). In addition, to investigate the expression of human # 414 mRNA in human tissues, use MultipleTissue
Northern Blot (manufactured by CLONTECH) Membrane was used.

A human # 414 probe was prepared. The 414 cDNA fragment obtained by the subtraction was used for PCR2.1 (INVITRO
GEN (made by GEN) as a template, and using the 414F1 primer (5′-CTG GAA GAA CTT CAC AGA (18mer) / SEQ ID NO:
9) and 414R1 primer (5'-CCA TGA CCC CAG TTA TCC
(18mer) / SEQ ID NO: 10). After extracting the PCR product with phenol / chloroform and ethanol precipitation,
About 600 bp confirmed by 3% low melting point agarose electrophoresis
PCR amplified DNA fragment from Wizard ^@ PCR Preps DNA Purificati
The probe was collected using on System (Promega) and used as a probe. Connect the probe to Prime-It II Random Primer Label
perform ^{3 2} P-labeled using a ling Kits (STRATAGENE Co.) and purified by NICK ^TM Column (Pharmacia).

The nylon membrane to which the RNA was transferred was placed on a hybridization buffer GMCbuffer (0.5 M NaH
₂ PO ₄ , 5 mM EDTA, 1% BSA, 7% SDS, 100 μg / ml salmon sperm D
NA, pH 7.2) treated at 65 ° C for 1 hour and labeled with ³² P
414 probe was added and incubated overnight at 65 ° C.
Remove the nylon membrane and add 2X SSC, 0.1% SDS solution at room temperature.
Twice for 5 minutes, 2X SSC, 0.1% SDS solution at 65 ° C for 15 minutes, 0.
5X SSC, 0.1% SDS solution at 65 ° C for 15 minutes, 0.2X SSC, 0.1
Washed with a% SDS solution at 65 ° C for 15 minutes. The washed nylon film was exposed to an X-ray film (manufactured by Kodak) at -80 ° C for 3 days. Expression levels were compared using 28S rRNA as an internal standard. Nurse cells Hs67, HSN27, S
Since expression was confirmed in K-LMS-1, human # 414 is presumed to be involved in the function of nurse cells (FIG. 5). Human heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,
When the expression of thymus, prostate, testis, ovary, large intestine, small intestine, and peripheral blood leukocytes in 16 types of tissues was examined, lung, heart, placenta, small intestine, large intestine, frontal line, testis, and ovaries were approximately 3 kb. A strong signal was detected in size, and weak expression was observed in other tissues (FIG. 6). No signal was detected in spleen, peripheral blood leukocytes, or liver. Further, expression was observed in all three types of human vascular endothelial cells, suggesting a relationship with arteriosclerosis (FIG. 7).

Example 5 Embedding MC / CAR (Pseudoem
peripolesis to MC / CAR) In order to analyze the relationship between the expression level of # 414 of the present invention and nurse cell activity, the MC / CAR-entrapping activities of the various cell lines used in the Northern blot analysis were compared. After incubating 2 × 10 ⁴ test cells / well shown in FIG. 5 at 37 ° C. for 2 days, 10 ⁶ cells / wel B-lymphoma-derived cell line MC / CAR was added thereto and mixed and cultured under the same conditions. After 6 hours, one test cell in which three or more MC / CAR cells had entered was found to be positive. At least 200 test cells were counted in one experimental system, and the number of positively embraced cells was expressed as a percentage. It has been previously confirmed that the B-lymphoma-derived cell line MC / CAR cells used in the experiment are embraced by nurse cells (Iwagami S. et al, J. Immunol., 15).
3: 2927, 1994). The results are shown in FIG. Nd shown in the figure
Indicates that no experiment was performed. However, another experiment has confirmed that at least about 50% of cells in the synovial nurse cell RA189SM contain lymphocytes (Takeuchi E., et al, Arthritis Rheum. 42: 221-228,199
9). According to FIG. 5, expression of # 414 was not observed in cells having a low lymphocyte entrapment activity, suggesting a relationship between the nurse cell entrapment activity and # 414.

[Example 6] Adhesion resistance test of # 414 to cyclosporin A Treatment with a drug such as cyclosporin A or FK506 inhibits calcineurin that regulates integrin-dependent cell adhesion And then F
Reduces tyrosine (Tyr) phosphorylation of intracellular proteins such as ocal Adhesion Kinase (FAK) and Paxillin. As a result, chemokinesis suppression in neutrophils (Bill Hendy
et al., Science, 258: 296-299, 1992; Moira A., et al., Na
Nature, 377: 75-79, 1995) and inhibition of cell adhesion in colon cancer cells Colo201 cells (Mohri T., et al., Cell Struct.
unct., 23: 255-264, 1998). Therefore, in order to confirm that # 414 of the present invention is involved in cell adhesion by a molecule other than integrin, an adhesion resistance test for cyclosporin A was performed. 1. Construction of Human # 414 Expression Vector The full length cDNA of human # 414 was cut out with restriction enzymes XhoI and NotI, and inserted into the SalI and NotI sites of the pDR2 vector-derived expression vector pDREF-Hyg vector. The resulting vector was transformed into E. coli DH5alpha, and the plasmid DNA was
Purified by AGEN Plasmid KIT (Qiagen).

2. Preparation of 293 / EBNA-Transformed Cells [0112] Human fibronectin (manufactured by GIBCO BRL) was added to a 6-well plate at a concentration of 10 mg / l in an amount of 1 ml and left at room temperature for 30 minutes. Thereafter, the cells were washed once with 2 ml of PBS, and the human embryonic kidney-derived cell line 293 / EBNA cells (ATCC No. CRL-1573) were treated with 6 wells.
l 10% FCS on plate with D-MEM (manufactured by Niken Biomedical Research Institute)
2 × 10 ⁵ cells were seeded per well using the added medium and cultured at 37 ° C. under 5% CO ₂ . 24 hours later, 2ml OP
Transformation was performed by adding TI-MEM I Reduced-Serum Medium. pDERF, pDERF- # 414 vector per well,
A mixture of 1 μg and 6 μl of Lipofectamine was added, and the mixture was cultured for 3 hours. Thereafter, the cells are washed with D-MEM, and DM
EM + 10% FCS culture solution was added and cultured. Next day, final concentration 200
mg / l of hygromycin and add another 1
After culturing for a week, the transformed cells were concentrated and selected. The cells were maintained in the presence of the above concentration of hygromycin.

3. Human # 414 cyclosporin A (CsA)
Adhesion resistance experiment on the above # 414 transformed cells were treated with trypsin, the cells were detached from the petri dish, centrifuged to separate the cells, suspended in the above-described hygromycin-added D-MEM + 10% FCS culture solution, and suspended in 6 We.
5 × 10 ⁵ cells were spread on each plate. At this time, cyclosporin A was added at a concentration of 15 mg / l, and the cells were cultured at 37 ° C. under 5% CO ₂ . Four days later, the number of cells adhered to the plate was counted randomly under a microscope for 10 fields. The results are shown in FIG. As a result, it was found that the decrease in cell adhesion ability due to cyclosporin A was partially restored in # 414 transformed cells.

Example 7 Preparation of pDREF- # 414myc Expression Vector Based on the cDNA sequence of # 414, primers required for constructing a chimeric gene of # 414 and myc tag were prepared. 414R19sal 5'-AAGTCGACGATAATGCAGATGACAGTACTG-3 '
(SEQ ID NO: 13) 414F01 5'-CTGGAAGAACTTCACAGA-3 '(SEQ ID NO: 1)
4)

The PCR template used was # 414 full-length cDNA. P
For the CR reaction, 100 ng of the template and the above primers at a final concentration of 1 pmol / μl were used, and Pfu polymerase (Invitrogen) was used for 1 hour.
0 U, attached Buffer and 2.5 mM dNTPs (final concentration 200 μM)
Was used to make the total volume 50 μl. The reaction is 94 ° C for 1 minute, 55 ° C for 1 minute.
For 15 minutes at 72 ° C. for 1 minute. The obtained PCR fragment was obtained in the same manner as in Example 1 (2) using the pCR2.1 vector (Invi
trogen) and the nucleotide sequence was determined.
In addition, # 414c at the HindIII restriction enzyme site within the reading frame for # 414
Recombinant with DNA, the stop codon was mutated to create # 414. This mutant # 414 was inserted into the pcDNA3.1 (+) myc / HisB vector so as to match the reading frame of the myc tag, and # 414m
yc was prepared. Check the nucleotide sequence of the junction, and
14myc was inserted into the pDREF vector to create pDREF- # 414myc.

[Example 8] Measurement of endocytosis activity by human # 414 by fluorescence microscopy pDREF- # 414my was added to human embryonic kidney-derived cell line 293 / EBNA-1 cells.
After transforming c, two days later, hygromycin at a final concentration of 0.2 μg / ml was added, and the cells were further cultured for one week, and the transformed cells were concentrated and selected. The transformed cells were seeded on a glass plate treated with human fibronectin at 5 × 10 ⁴ cells at a time, and D-MEM +
A 10% FCS culture solution was added, and the cells were cultured for 24 hours. Then final concentration 10
An anti-myc antibody was added to a concentration of μg / ml. 4 ℃ or 37 ℃
For 2 hours, and then washed three times with D-MEM + 10% FCS.
After fixation with 3.7% formalin / PBS for 10 minutes, the cells were permealized with 0.5% Triton / PBS. After that, D-MEM + 10%
After washing twice with FCS, the final concentration of FITC-labeled anti-mouse IgG was 2 μg / m
l and left for 1 hour. Furthermore, after washing twice with D-MEM + 10% FCS, the cells were observed with a fluorescence microscope (FIG. 9A).

As a result of the microscopic examination, a typical stained image obtained is shown in FIG. 9A. At 4 ° C., most of the stained cells showed an image in which only the cell surface was stained as shown in the figure. Furthermore, at 37 ° C., most of the stained cells showed granular stained images inside the cells. No staining was observed in the # 414-expressing cells used as a control. These results suggested that in the # 414myc-transformed cells, the anti-myc antibody bound to # 414myc was taken up into intracellular granular organelles by endocytosis. At 4 ° C., the mechanism of endocytosis does not function due to low temperature, so that it was not taken up into cells and only the cell surface was stained. This result also suggests that # 414 is expressed on the cell surface.

[0118] Example 9 was used ¹⁰⁶ respectively quantification the # 414Myc and control cells as # 414 transformed cells by FACS endocytic activity of human # 414. The cells were detached from the culture dish with PBS containing EDTA at a final concentration of 2 mM, and the cells were collected by centrifugation and suspended in D-MEM + 10% FCS to a concentration of 5 × 10 ⁶ / ml. Anti-myc was added to the cell suspension at a final concentration of 10 μg / ml as described above.
The antibody was added and left at 4 ° C. or 37 ° C. for 2 hours. Then perform formalin fixation and permealize treatment as above,
A secondary antibody, anti-mouse IgG, was added at a final concentration of 2 μg / ml and left for 1 hour. After washing twice with 1% BSA / PBS, the results of FACS analysis are shown in FIG. 9B. At 37 ° C, the # 414myc-transformed cells showed a 4.2-fold increase in the X-axis mean (X-mean) compared to the control # 414-transformed cells. In the experiment at 37 ° C., the background of the control was set to 0.5% or less, and the frequency of positive cells in # 414myc-expressing cells was analyzed based on the background at that time, and it was 37.0%.

At 4 ° C., the average of the X axis was 1.2 times, but the frequency of positive cells increased from 0.49% to 4.56%. From the above results, the anti-myc antibody was bound to the cells,
At 37 ° C., the fluorescence intensity was increased about 4 times at 37 ° C.
After fixing and expressing permealize # 414myc-expressing cells and treating and staining with anti-myc antibody at 37 ° C or 4 ° C, FACS analysis showed no difference between the two. Does not appear to be significantly different. Therefore, the above results are considered to indicate that the anti-myc antibody was accumulated in the cells by endocytosis. Considering that the gene of the present invention is expressed in vascular endothelial cells, it is considered that the protein encoded by the gene of the present invention is involved in the uptake of a specific ligand from the vascular lumen by endocytosis. Can be

[0120] Example 10 fluorescence microscope according to # 414 of the identification above # 414 transformed cells Ligand seeded 10 ⁵ a glass plate with human fibronectin coated, 24-hour D-MEM + 10% FC
Cultured in S. Various sugar chains [N-acetylgalactosamine
(GalNAc), D-glucose (Glu), mannose (Man)] bound to polyacrylamide, and further labeled with biotin (GallNAc-BP or generically referred to as sugar chain-BP) (from Seikagaku Corporation). Was dissolved in water to a concentration of 1 mg / ml. This GalNAc-BP and an equal amount of FITC-labeled avidin (manufactured by Amersham-pharmacia) were mixed and allowed to stand at room temperature for 1 hour. As a result, biotin and avidin bind to each other,
-BP probe is FITC labeled.

This mixed solution was treated with the above # 414 expression 293 / EBNA-1.
Carbohydrate-BP was added to cells or 293 / EBNA-1 cells expressing macrophage lectin as a positive control or 293 / EBNA-1 cells expressing only vector as a negative control.
To a final concentration of 50 μg / ml in D-MEM + 10% FCS.
The cells were cultured at 37 ° C. for 3 hours. Thereafter, the plate was washed three times with D-MEM + 10% FCS, and further fixed with 3.7% formalin / PBS for 10 minutes. Further, it was washed once with D-MEM + 10% FCS, and the result of observation with a fluorescence microscope is shown in FIG. Positive control is GalNAc-BP
Only incorporation is shown. As a result, apparently GalNAc-BP
Only cells were taken into the cells, and a granular staining image was observed. Other sugar chains such as Glu and Man were not incorporated at all.

Example 11 Quantitative Analysis and Identification of Ligand of # 414 by FACS 5 × 10 ⁶ transformants of the above-mentioned # 414 were seeded on a 10 cm dish and cultured in D-MEM + 10% FCS for 24 hours. . Above, sugar chain-BP probe, N-acetylgalactosamine GallNAc-BP + FITC
Labeled avidin (Amersham-pharmacia) mixed solution
# 414-expressing 293 / EBNA-1 cells, or 293 / EBNA-1 cells expressing macrophage lectin as a positive control or 293 / EBNA-1 cells expressing the vector alone as a negative control
Sugar chain-BP is added to EBNA-1 cells to a final concentration of 50 μg / ml, and cultured in an Eppendorf tube at a cell concentration of 5 × 10 ⁶ / ml in D-MEM + 10% FCS at 37 ° C. for 1 hour did. Then D-ME
Wash 3 times with M + 10% FCS, then add 1% with 3.7% formalin / PBS
Fixed for 0 minutes. Further, it was washed twice with PBS + 10% FCS and analyzed by FACS. The result is shown in FIG. The positive control is Ga
By binding or incorporating lNAc-BP, the average fluorescence intensity on the X-axis is about 5.0 compared to vector-only transformed cells.
Doubled. # 414 similarly rose about 2.0 times. From these results, it is clear that GalNAc-BP binds to or is incorporated into the # 414 gene product.

[Example 12] Lectin region (# 414Lec) of # 414 gene and SEAP (secretory alkaline phosphatase)
To clone only the lectin region of the 414 gene, PCR was performed using primers (F10Bam and R10Xba).
Amplified by With this primer, a region encoding 153 amino acid residues at the C-terminal side, which is a lectin region of # 414 protein, is amplified. F10Bam 5'-GGATCCGGCCCATCAGGAGCGGTG-3 '(SEQ ID NO: 15) R10Xba 5'-TCTAGATAATGCAGATGACAGTAC-3' (SEQ ID NO: 16)

After confirming the nucleotide sequence, the pDisplay vector (Invi
trogen). With this, 414Lec
The IgG signal sequence in the pDisplay vector is linked in-frame to form a secreted 414Lec. And then
pDRAP vector (pDREF vector into which a defective alkaline phosphatase lacking the signal sequence of SEAP has been inserted in advance, and His-Tag is added to the C-terminal of alkaline phosphatase.
Is attached. ) Insert into Xba I and secrete # 414Lec C
A chimeric gene expression vector in which SEAP was connected in-frame at the end was prepared (referred to as pDRAP-414Lec vector). This vector was transformed into 293 / EBNA cells (D-MEM / 10% FCS), and after 3 days, the culture supernatant of the cells was collected and SEAP activity was measured. The activity was measured using the Great EscAPe kit (Clontech
Was used.

As a result, almost the same activity was observed as when only SEAP was expressed per unit culture supernatant in the culture supernatant. Using a nickel resin purchased from Qiagen from the supernatant, the His-Tag was bound to the column as usual, and after washing three times, the chimeric protein (# 414 lectin region (# 414Lec) and alkaline phosphatase (AP)) was used. # 414
Called Lec-AP. ) Was purified by eluting with 250 mM imidazole.

AP or # 414Lec-AP purified protein 10
The μl was subjected to SDS-PAGE and subjected to Coomassie staining. As a result, two bands were detected at a size of about 75 kD for the AP protein (lane 1). A specific band was detected (lane 2) (FIG. 12). Since the expected molecular weight of # 414Lec-AP protein is about 70 kD, it is considered that the molecular weight has increased due to glycosylation and the like. As a result, the # 414Lec-AP protein could be purified with almost 100% purity. Since the specific activities of the alkaline phosphatase of the purified # 414Lec-AP protein and the AP protein were almost the same, it is considered that the chimera did not affect the activity of the AP region.

Example 13 Ga by purified # 414-Lec
lNAc-gel binding assay At a final concentration of 100 μg / ml, purified # 414Lec-AP protein or 10 μg each of AP protein as a negative control
The mixture was mixed with 20 μl of N-acetylgalactosamine-immobilized gel (manufactured by EY laboratories) in a binding buffer (D-MEM / 0.2% BSA / 4 mM Ca ^{2 +} buffer) and stirred at room temperature for 1 hour with constant stirring. And the mixture was allowed to stand while the gel did not precipitate. Then, it was washed three times with 1 ml of the same buffer. For washing, centrifuge at 15,000 rpm for 10 seconds after adding the washing solution,
GalNAc-gel was precipitated, and the supernatant was drawn off with a pipetman or the like. In the inhibition experiments, 50 mM EDTA or 100 mM N-acetylgalactosamine, D-galactose, N-acetylglucosamine or mannose or 1 mg / ml mannan, dextran,
Add fucoidan or chondroitin sulfate A (all from SIGMA) to the final concentrations shown, and add # 414
It was examined whether the binding between Lec-AP and GalNAc-gel was inhibited (FIG. 13). After washing, the AP activity bound or not bound to the gel was measured according to a standard method.

As a result, about 10% of the added AP activity was GalNAc.
-Bound to gel, but not AP alone. Therefore, it was found that the protein was bound to the GalNAc-gel via the # 414Lec region. Furthermore, by the inhibition experiment,
More than 95% of the binding of # 414Lec-AP was inhibited by 50 mM EDTA, 100 mM N-acetylgalactosamine and D-galactose. Since the gel was washed three times, little EDTA and monosaccharides are likely to remain. In another experiment, since the AP activity was not inhibited in the presence of 100 mM of the above-mentioned various monosaccharides, it was concluded that the AP activity in the gel quantified the amount of # 414Lec-AP bound to the gel. From this result, it was found that # 414Lec-AP specifically binds to N-acetylgalactosamine and D-galactose.

Example 14 Measurement of 50% Inhibitory Concentration (IC ₅₀ ) of Monosaccharide by N-Acetylgalactosamine for Binding of # 414Lec-AP to GalNAc-Gel Add the sugar N-acetylgalactosamine to the binding assay
The concentration of N-acetylgalactosamine to be 50% was determined. As a result, IC with about N-acetylgalactosamine
₅₀ was found to be about 3 mM (FIG. 14).

[Example 15] Binding of # 414Lec to cells T lymphoma l derived from human Acute Lymphoblastic leukemia
ine is Jurkat, Molt17 and B lymphoma line
2 × 10 ⁶ BALL-1 cells were added to 0.5 ml of binding buffer (D-
MEM, 20mM-HEPES (pH7.5), 0.2mM BSA and 4mM CaCl ₂
Or 4mM EDTA) in # 414Lec-AP or SEAP 1
The mixture was stirred at room temperature for 1 hour and allowed to stand.
Then, 3 ml with 1 ml of the above binding buffer containing 4 mM CaCl ₂
Washed twice. Washing was performed by centrifuging the cells at 3,000 rpm for 1 minute and pelleting down. Thereafter, the remaining alkaline phosphatase activity of the cells was quantified. Figure 1 shows the results
It is shown in FIG. As a result, # 414Lec bound to Jurkat and BALL-1 in a calcium-dependent manner, but did not bind to Molt17 cells. Binding to Jurkat or BALL-1 was almost the same, and calcium chelation by EDTA reduced binding activity by 20-30 fold.

[0131]

According to the present invention, a novel scavenger receptor-like protein is provided. Since the scavenger receptor-like protein of the present invention is highly expressed in nurse-like cells including the synovial membrane of human RA patients,
It may be deeply involved in autoimmune symptoms in RA and the like. Therefore, the diagnosis of autoimmune diseases like RA,
It is useful as a target molecule for prevention and treatment. In addition, since it may support osteoclast differentiation and activation, it may be closely related to the pathology of arthritis in RA. If bone destruction can be inhibited by inhibiting the function of nurse cells, it may be possible to control the pathology of arthritis in RA. Furthermore, considering that nurse cells produce inflammatory cytokines, it can be said that the scavenger receptor-like protein of the present invention is extremely important as a target molecule for RA treatment. The scavenger receptor-like protein according to the present invention is also expressed in vascular endothelial cells. Since vascular endothelium is a place where atherosclerotic lesions are formed, it is possible that the scavenger receptor-like protein of the present invention plays an important role in atherosclerosis. Therefore, the scavenger receptor-like protein of the present invention or a gene encoding the same is also useful as a target molecule for preventing or treating arteriosclerosis. That is, the protein according to the present invention, or the gene encoding the same,
It can be used as a target molecule for screening for a substance that inhibits its function. The substance obtained by the screening based on the present invention can be used as a candidate substance for a prophylactic or therapeutic agent for RA or arteriosclerosis.

Alternatively, if the relationship between nurse cells and cancer cells found in primary and metastatic foci of cancer can be explained through the protein of the present invention, the function of cancer can be controlled by adjusting the function. Can be expected. Therefore, the present invention can be expected to be useful in prevention and treatment of cancer.

On the other hand, cell adhesion activity was found in the scavenger receptor-like protein of the present invention. Although its adhesive activity is weaker than that of SRA, it shows adhesive activity even in the presence of cyclosporin A, suggesting that it adheres by a different mechanism from integrin.
It was also presumed that the coiled-coil region, collagen region and C-type lectin region of the scavenger receptor-like protein functioned for the adhesion. In particular, adhesion through the C-type lectin region, that is, adhesion through sugar chains, is characterized by mediating relatively “weak” adhesion. Such adhesion is an early stage of cell adhesion, especially a step of mutual discrimination between cells. Have been suggested to play an important role. In general, it has been reported that integrins are also expressed in fibroblasts having no nurse cell function and function in cell adhesion of these cells. However, the scavenger receptor-like protein of the present invention "embraces" lymphocytes (Pseudoemperer
The scavenger receptor-like protein of the present invention is expressed in cells having the capability of ipolesis, that is, nurse cells.
oemperipolesis). Therefore, the cell adhesion activity of the scavenger receptor-like protein of the present invention may be acting in a direction to enhance various disease states caused by lymphocytes. From these facts, if the cell adhesion activity of nurse cells can be suppressed through the inhibition of the function of the scavenger receptor-like protein of the present invention, inhibition of costimulation signal transmission to lymphocytes and inhibition of antigen presentation can be realized. It can be expected to lead to control of the disease state. The present invention also provides a method for screening a scavenger receptor-like protein, its ligand, and a compound that inhibits binding to the ligand. Compounds that inhibit the binding to ligands provided by these screening methods are useful as therapeutic agents in various disease states based on the above-mentioned systems.

[0134]

[Sequence List] SEQUENCE LISTING <110> Shionogi & Co., Ltd. <120> Scavenger receptor like protein <130> S4-102DP1 <140> <141> <150> JP 2000-90772 <151> 2000-03-27 <160> 16 <170> PatentIn Ver. 2.1 <210> 1 <211> 2262 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (34) .. (2259) <400> 1 ctccccggcc ggcggtgcgt ccccacggtc acc atg aaa gac gac ttc gca gag 54 Met Lys Asp Asp Phe Ala Glu 15 gag gag gag gtg caa tcc ttc ggt tac aag cgg ttt ggt att cag gaa 102 Glu Glu Glu Glu Glu P Gly Ile Gln Glu 10 15 20 gga aca caa tgt acc aaa tgt aaa aat aac tgg gca ctg aag ttt tct 150 Gly Thr Gln Cys Thr Lys Cys Lys Asn Asn Trp Ala Leu Lys Phe Ser 25 30 35 atc ata tta tta tac att ttg tgt gcc ttg cta aca atc aca gta gcc 198 Ile Ile Leu Leu Tyr Ile Leu Cys Ala Leu Leu Thr Ile Thr Val Ala 40 45 50 55 att ttg gga tat aaa gtt gta gag aaa atg gac aat gtc aca ggt ggc 246 Ile Leu Gly Tyr Lys Val Val Glu Lys Met Asp Asn Val Thr Gly Gly 60 65 70 atg gaa aca tct cgc caa acc tat gat gac aag ct c aca gca gtg gaa 294 Met Glu Thr Ser Arg Gln Thr Tyr Asp Asp Lys Leu Thr Ala Val Glu 75 80 85 agt gac ctg aaa aaa tta ggt gac caa act ggg aag aaa gct atc agc 342 Ser Asp Leu Lys Lys Leu Gly Asp Gln Thr Gly Lys Lys Ala Ile Ser 90 95 100 acc aac tca gaa ctc tcc acc ttc aga tca gac att cta gat ctc cgt 390 Thr Asn Ser Glu Leu Ser Thr Phe Arg Ser Asp Ile Leu Asp Leu Arg 105 110 115 cag caa ctt cgt gag att aca gaa aaa acc agc aag aac aag gat acg 438 Gln Gln Leu Arg Glu Ile Thr Glu Lys Thr Ser Lys Asn Lys Asp Thr 120 125 130 135 ctg gag aag tta cag gcg agc ggg gat gct ctg gtg gac agg cagt 486 Leu Glu Lys Leu Gln Ala Ser Gly Asp Ala Leu Val Asp Arg Gln Ser 140 145 150 caa ttg aaa gaa act ttg gag aat aac tct ttc ctc atc acc act gta 534 Gln Leu Lys Glu Thr Leu Glu Asn Asn Ser Phe Leu Ile Thr Thr Val 155 160 165 aac aaa acc ctc cag gcg tat aat ggc tat gtc acg aat ctg cag caa 582 Asn Lys Thr Leu Gln Ala Tyr Asn Gly Tyr Val Thr Asn Leu Gln Gln 170 175 180 gat acc agc gtg ctc cag ggc ag ctg cag aac caa atg tat tct cat 630 Asp Thr Ser Val Leu Gln Gly Asn Leu Gln Asn Gln Met Tyr Ser His 185 190 195 aat gtg gtc atc atg aac ctc aac aac ctg aac ctg acc cag gtg cag 678 Asn Val Val Ile Met Asn Leu Asn Asn Leu Asn Leu Thr Gln Val Gln 200 205 210 215 cag agg aac ctc atc acg aat ctg cag cgg tct gtg gat gac aca agc 726 Gln Arg Asn Leu Ile Thr Asn Leu Gln Arg Ser Val Asp Asp Thr Ser 220 225 230 cag gct atc cag cga atc aag aac gac ttt caa aat ctg cag cag gtt 774 Gln Ala Ile Gln Arg Ile Lys Asn Asp Phe Gln Asn Leu Gln Gln Val 235 240 245 ttt ctt caa gcc aag aag gac acg gat tgg ctg agt gag aaa cag 822 Phe Leu Gln Ala Lys Lys Asp Thr Asp Trp Leu Lys Glu Lys Val Gln 250 255 260 agc ttg cag aca ctg gct gcc aac aac tct gcg ttg gcc aaa gcc aac 870 Ser Leu Gln Thr Leu Ala Ala Asn Asn Ser Ala Leu Ala Lys Ala Asn 265 270 275 aac gac acc ctg gag gat atg aac agc cag ctc aac tca ttc aca ggt 918 Asn Asp Thr Leu Glu Asp Met Asn Ser Gln Leu Asn Ser Phe Thr Gly 280 285 290 290 295 cag atg gag aac acc act atc tct caa gcc aac gag cag aac ctg 966 Gln Met Glu Asn Ile Thr Thr Ile Ser Gln Ala Asn Glu Gln Asn Leu 300 305 310 aaa gac ctg cag gac tta cac aaa gat gca gag aat aga aca gcc atc 1014 Lys Asp Leu Gln Asp Leu His Lys Asp Ala Glu Asn Arg Thr Ala Ile 315 320 325 aag ttc aac caa ctg gag gaa cgc ttc cag ctc ttt gag acg gat att 1062 Lys Phe Asn Gln Leu Glu Glu Arg Phe Gln Leu Phe Glu Thr As 335 340 gtg aac atc att agc aat atc agt tac aca gcc cac cac ctg cgg acg 1110 Val Asn Ile Ile Ser Asn Ile Ser Tyr Thr Ala His His Leu Arg Thr 345 350 355 ctg acc agc aat cta aat gaa gtc agg acc act tc aca gat acc ctt 1158 Leu Thr Ser Asn Leu Asn Glu Val Arg Thr Thr Cys Thr Asp Thr Leu 360 365 370 375 acc aaa cac aca gat gat ctg acc tcc ttg aat aat acc ctg gcc aac 1206 Thr Lys His Thr Asp Asp Leu Thr Ser Leu Asn Asn Thr Leu Ala Asn 380 385 390 atc cgt ttg gat tct gtt tct ctc agg atg caa caa gat ttg atg agg 1254 Ile Arg Leu Asp Ser Val Ser Leu Arg Met Gln Gln Asp Leu Met Arg 395 400 405 tcg agg tta gac act gaa gta gcc aac tta tca gtg att atg gaa gaa 1302 Ser Arg Leu Asp Thr Glu Val Ala Asn Leu Ser Val Ile Met Glu Glu 410 415 420 atg aag cta gta gac tcc aag cat ggt cag ctc atc aag aat ttt aca 1350 Met Lys Leu Val Asp Ser Lys His Gly Gln Leu Ile Lys Asn Phe Thr 425 430 435 ata cta caa ggt cca ccg ggc ccc agg ggt cca aga ggt gac aga gga 1398 Ile Leu Gln Gly Pro Pro Gly Pro Arg Gly Pro Arg Gly Asp Arg Gly 440 445 450 455 tcc cag gga ccc cct ggc cca act ggc aac aag gga cag aaa gga gag 1446 Ser Gln Gly Pro Pro Gly Pro Thr Gly Asn Lys Gly Gln Lys Gly Glu 460 465 470 aag ggg gag cct gga cca cct ggc cct gcg ggt gag aga ggc cca att 1494 Lys Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Glu Arg Gly Pro Ile 475 480 485 gga cca gct ggt ccc ccc gga gag cgt ggc ggc aaa gga tct aaa ggc 1542 Gly Gly Pro Pro Gly Glu Glu Arg Gly Gly Lys Gly Ser Lys Gly 490 495 500 tcc cag ggc ccc aaa ggc tcc cgt ggt tcc cct ggg aag ccc ggc cct 1590 Ser Gln Gly Pro Lys Gly Ser Arg Gly Ser Pro Gly Lys Pro Gly P ro 505 510 515 cag ggc ccc agt ggg gac cca ggc ccc ccg ggc cca cca ggc aaa gag 1638 Gln Gly Pro Ser Gly Asp Pro Gly Pro Pro Gly Pro Pro Gly Lys Glu 520 525 530 535 gga ctc ccc ggc cct cag ggc c ggc ttc cag gga ctt cag ggc 1686 Gly Leu Pro Gly Pro Gln Gly Pro Pro Gly Phe Gln Gly Leu Gln Gly 540 545 550 acc gtt ggg gag cct ggg gtg cct gga cct cgg gga ctg cca ggc ttg 1734 Thr Val Gly Glu Val Pro Gly Pro Arg Gly Leu Pro Gly Leu 555 560 565 cct ggg gta cca ggc atg cca ggc ccc aag ggc ccc ccc ggc cct cct 1782 Pro Gly Val Pro Gly Met Pro Gly Pro Lys Gly Pro Pro Gly Pro Pro 570 575 575 580 ggc cca tca gga gcg gtg gtg ccc ctg gcc ctg cag aat gag cca acc 1830 Gly Pro Ser Gly Ala Val Val Pro Leu Ala Leu Gln Asn Glu Pro Thr 585 590 595 ccg gca ccg gag gac aat ggc tgc ccg cct cac tgg ag aca 1878 Pro Ala Pro Glu Asp Asn Gly Cys Pro Pro His Trp Lys Asn Phe Thr 600 605 610 gac aaa tgc tac tat ttt tca gtt gag aaa gaa att ttt gag gat gca 1926 Asp Lys Cys Tyr Tyr Phe Ser Val Glu Ly s Glu Ile Phe Glu Asp Ala 620 625 630 aag ctt ttc tgt gaa gac aag tct tca cat ctt gtt ttc ata aac act 1974 Lys Leu Phe Cys Glu Asp Lys Ser Ser His Leu Val Phe Ile Asn Thr 635 640 645 645 aga gag gaa cag caa tgg ata aaa aaa cag atg gta ggg aga gag agc 2022 Arg Glu Glu Gln Gln Trp Ile Lys Lys Gln Met Val Gly Arg Glu Ser 650 655 660 cac tgg atc ggc ctc aca gac tca gag cgt gaa aat tgaa tag aag Trp Ile Gly Leu Thr Asp Ser Glu Arg Glu Asn Glu Trp Lys Trp 665 670 675 ctg gat ggg aca tct cca gac tac aaa aat tgg aaa gct gga cag ccg 2118 Leu Asp Gly Thr Ser Pro Asp Tyr Lys Asn Trp Lys Ala Gly Gln Pro 680 685 690 695 gat aac tgg ggt cat ggc cat ggg cca gga gaa gac tgt gct ggg ttg 2166 Asp Asn Trp Gly His Gly His Gly Pro Gly Glu Asp Cys Ala Gly Leu 700 705 710 att tat gct ggg cag tgg aac gat ttc caa tgt gaa gac gtc aat aac 2214 Ile Tyr Ala Gly Gln Trp Asn Asp Phe Gln Cys Glu Asp Val Asn Asn 715 720 725 ttc att tgc gaa aaa gac agg gag aca gta ctg tca tct gca tta taa 2262 Plu Ile Cy s Asp Arg Glu Thr Val Leu Ser Ser Ala Leu 730 735 740 <210> 2 <211> 742 <212> PRT <213> Homo sapiens <400> 2 Met Lys Asp Asp Phe Ala Glu Glu Glu Glu Val Gln Ser Phe Gly Tyr 1 5 10 15 Lys Arg Phe Gly Ile Gln Glu Gly Thr Gln Cys Thr Lys Cys Lys Asn 20 25 30 Asn Trp Ala Leu Lys Phe Ser Ile Ile Leu Leu Tyr Ile Leu Cys Ala 35 40 45 Leu Leu Thr Ile Thr Val Ala Ile Leu Gly Tyr Lys Val Val Glu Lys 50 55 60 Met Asp Asn Val Thr Gly Gly Met Glu Thr Ser Arg Gln Thr Tyr Asp 65 70 75 80 Asp Lys Leu Thr Ala Val Glu Ser Asp Leu Lys Lys Leu Gly Asp Gln 85 90 95 Thr Gly Lys Lys Ala Ile Ser Thr Asn Ser Glu Leu Ser Thr Phe Arg 100 105 110 Ser Asp Ile Leu Asp Leu Arg Gln Gln Leu Arg Glu Ile Thr Glu Lys 115 120 125 Thr Ser Lys Asn Lys Asp Thr Leu Glu Lys Leu Gln Ala Ser Gly Asp 130 135 140 Ala Leu Val Asp Arg Gln Ser Gln Leu Lys Glu Thr Leu Glu Asn Asn 145 150 155 160 Ser Phe Leu Ile Thr Thr Val Val Asn Lys Thr Leu Gln Ala Tyr Asn Gly 165 170 175 Tyr Val Thr Thr Asn Leu Gln Gln Asp Thr Ser Val Leu Gln Gly Asn Leu 1 80 185 190 Gln Asn Gln Met Tyr Ser His Asn Val Val Ile Met Asn Leu Asn Asn 195 200 205 Leu Asn Leu Thr Gln Val Gln Gln Arg Asn Leu Ile Thr Asn Leu Gln 210 215 220 Arg Ser Val Asp Asp Thr Ser Gln Ala Ile Gln Arg Ile Lys Asn Asp 225 230 235 240 Phe Gln Asn Leu Gln Gln Val Phe Leu Gln Ala Lys Lys Asp Thr Asp 245 250 255 Trp Leu Lys Glu Lys Val Gln Ser Leu Gln Thr Leu Ala Ala Asn Asn 260 265 270 270 Ser Ala Leu Ala Lys Ala Asn Asn Asp Thr Leu Glu Asp Met Asn Ser 275 280 285 Gln Leu Asn Ser Phe Thr Gly Gln Met Glu Asn Ile Thr Thr Ile Ser 290 295 300 Gln Ala Asn Glu Gln Asn Leu Lys Asp Leu Gln Asp Leu His Lys Asp 305 310 315 320 Ala Glu Asn Arg Thr Ala Ile Lys Phe Asn Gln Leu Glu Glu Arg Phe 325 330 335 Gln Leu Phe Glu Thr Asp Ile Val Asn Ile Ile Ser Asn Ile Ser Tyr 340 345 350 Thr Ala His His Leu Arg Thr Leu Thr Ser Asn Leu Asn Glu Val Arg 355 360 365 Thr Thr Cys Thr Asp Thr Leu Thr Lys His Thr Asp Asp Leu Thr Ser 370 375 380 Leu Asn Asn Thr Leu Ala Asn Ile Arg Leu Asp Ser Val Ser Leu Arg 385390 395 400 Met Gln Gln Asp Leu Met Arg Ser Arg Leu Asp Thr Glu Val Ala Asn 405 410 415 Leu Ser Val Ile Met Glu Glu Met Lys Leu Val Asp Ser Lys His Gly 420 425 430 Gln Leu Ile Lys Asn Phe Thr Ile Leu Gln Gly Pro Pro Gly Pro Arg 435 440 445 Gly Pro Arg Gly Asp Arg Gly Ser Gln Gly Pro Pro Gly Pro Thr Gly 450 455 460 Asn Lys Gly Gln Lys Gly Glu Lys Gly Glu Pro Gly Pro Pro Gly Pro 465 470 475 480 Ala Gly Glu Arg Gly Pro Ile Gly Pro Ala Gly Pro Pro Gly Glu Arg 485 490 495 Gly Gly Lys Gly Ser Lys Gly Ser Gln Gly Pro Lys Gly Ser Arg Gly 500 505 510 510 Ser Pro Gly Lys Pro Gly Pro Gln Gly Pro Ser Gly Asp Pro Gly Pro 515 520 525 Pro Gly Pro Pro Gly Lys Glu Gly Leu Pro Gly Pro Gln Gly Pro Pro 530 535 540 Gly Phe Gln Gly Leu Gln Gly Thr Val Val Gly Glu Pro Gly Val Pro Gly 545 550 555 560 Pro Arg Gly Leu Pro Gly Leu Pro Gly Val Pro Gly Met Pro Gly Pro 565 570 575 Lys Gly Pro Pro Gly Pro Pro Gly Pro Ser Gly Ala Val Val Pro Leu 580 585 590 Ala Leu Gln Asn Glu Pro Thr Pro Ala Pro Glu Asp Asn Gly Cys Pro 595 600 605 Pro His Trp Lys Asn Phe Thr Asp Lys Cys Tyr Tyr Phe Ser Val Glu 610 615 620 lys Glu Ile Phe Glu Asp Ala Lys Leu Phe Cys Glu Asp Lys Ser Ser 625 630 635 640 His Leu Val Phe Ile Asn Thr Arg Glu Glu Gln Gln Trp Ile Lys Lys 645 650 655 Gln Met Val Gly Arg Glu Ser His Trp Ile Gly Leu Thr Asp Ser Glu 660 665 670 Arg Glu Asn Glu Trp Lys Trp Leu Asp Gly Thr Ser Pro Asp Tyr Lys 675 680 685 Asn Trp Lys Ala Gly Gln Pro Asp Asn Trp Gly His Gly His Gly Pro 690 695 700 Gly Glu Asp Cys Ala Gly Leu Ile Tyr Ala Gly Gln Trp Asn Asp Phe 705 710 715 715 720 Gln Cys Glu Asp Val Asn Asn Phe Ile Cys Glu Lys Asp Arg Glu Thr 725 730 735 Val Leu Ser Ser Ala Leu 740 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 3 cagctccaca acctacatca ttccgt 26 <210> 4 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 4 acggaatgat gt 12 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 5 gtccatcttc tctctgagac tctggt 26 <210> 6 <211> 12 <212 > DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 6 accagagtct ca 12 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> < 223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 7 aagaaaaacc tgctgcagat tttg 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 8 gtcgttcttg attcgctgga tagc 24 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 9 ctggaagaac ttcacaga 18 <210 > 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synth esized Primer Sequence <400> 10 ccatgacccc agttatcc 18 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 11 cagatggaga acatcacc 18 < 210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 12 cttcatttct tccataatca 20 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 13 aagtcgacga taatgcagat gacagtactg 30 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 14 ctggaagaac ttcacaga 18 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence < 400> 15 ggatccggcc catcaggagc ggtg 24 <210> 16 <211> 24 <212> DNA <213> Artificial Sequenc e <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 16 tctagataat gcagatgaca gtac 24

[Brief description of the drawings]

[Figure 1] Human scavenger receptor-like protein # 4
It is a figure which shows the base sequence of 14cDNA.

FIG. 2 is a view showing an amino acid sequence of # 414 protein.

FIG. 3 is a view showing a comparison of the amino acid sequences of # 414 protein and SRA-I protein. “*” Indicates the same, “:” indicates a highly similar amino acid, and “.” Indicates a similar amino acid.

FIG. 4 is a view showing a domain structure of # 414 protein.

FIG. 5 is a photograph showing the results of Northern hybridization in which the expression of # 414 in various cultured cell lines was analyzed.

FIG. 6 is a photograph showing the results of Northern hybridization in which the expression of # 414 in various tissues was analyzed.

FIG. 7 is a photograph showing the results of Northern hybridization for analyzing the expression of # 414 in a vascular endothelial cell line.

FIG. 8 is a graph showing the results of an adhesion resistance test for cyclosporin A of # 414. The vertical axis indicates the number of adherent cells counted in 10 visual fields.

FIG. 9A is a photograph showing the result of measuring the endocytosis activity of # 414 using a fluorescence microscope. The upper row shows # 414 expression cells used as controls, the lower row shows # 414myc
Transformed cells. B is a graph showing the result of analyzing the endocytosis activity of # 414 by FACS. The upper row shows # 414 expression cells used as controls, the lower row shows
# 414myc transformed cells.

FIG. 10 is a photograph showing the result of measuring the incorporation of various sugar chains in # 414 using a fluorescence microscope. The left column shows the vector alone as a negative control.
EBNA-1 cells, middle row: # 414-expressing 293 / EBNA-1 cells, right row: positive control macrophage lectin-expressing 293 / EBNA-1 cells.

FIG. 11 is a graph showing the results of analyzing the incorporation of sugar chains at # 414 by FACS. The upper row shows 293 / EBNA-1 cells expressing macrophage lectin as a positive control, and the lower row shows 293 / EBNA-1 cells expressing # 414.

FIG. 12: SDS- of purified protein of AP or # 414Lec-AP
4 is a photograph showing a result of PAGE. Lane 1 is the AP protein and lane 2 is the # 414Lec-AP protein.

FIG. 13 is a graph showing the degree of inhibition of binding between # 414Lec-AP and GalNAc-gel by adding various inhibitors. The vertical axis is # 4
The binding amount (%) to 14Lec-AP is shown, and the horizontal axis represents the type of inhibitor.

FIG. 14: N for binding of # 414Lec-AP to GalNAc-gel
It is a graph which shows the result of measurement of 50% inhibitory concentration (IC50) of -acetylgalactosamine.

FIG. 15 is a graph showing the results of measuring the binding activity of # 414Lec-AP to various cells. The vertical axis represents the relative activity (%) of alkaline phosphatase remaining on the cells relative to the activity of added alkaline phosphatase. The horizontal axis indicates the cell type. For each cell, the binding in the absence of EDTA is represented by a black column, and the binding in the presence is represented by a white column.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 3/10 A61P 9/14 4H045 9/10 101 25/28 9/14 27/02 25/28 29 / 00 101 27/02 35/00 29/00 101 37/00 35/00 C07K 14/705 37/00 16/28 C07K 14/705 C12N 1/15 16/28 1/19 C12N 1/15 1/21 1 / 19 C12P 21/02 C 1/21 21/08 5/10 C12Q 1/00 Z C12P 21/02 C12N 15/00 ZNAA 21/08 A61K 37/02 C12Q 1/00 C12N 5/00 A B F term ( Reference) 4B024 AA01 AA11 BA61 BA63 CA01 HA01 HA11 HA17 4B063 QA01 QA18 QA19 QQ02 QQ42 QQ79 QQ96 QR32 QR48 QS07 QS33 QS36 QX01 4B064 AG20 AG27 CA01 CA10 CA19 CC24 DA06 DA13 4B065 AA93A01 CA01 A08A14 A08A14 BA08 BA13 BA22 BA23 CA20 CA23 CA25 DC50 NA14 ZA162 ZA332 ZA442 ZA452 ZB072 ZB152 ZB262 ZC352 4H045 AA10 AA11 BA10 CA40 DA50 DA76 EA23 EA50 FA74

Claims (29)

[Claims] 1. A protein comprising the amino acid sequence of SEQ ID NO: 2. 2. The amino acid sequence of SEQ ID NO: 2 which comprises an amino acid sequence in which one or several amino acids have been substituted, deleted, added and / or inserted. A protein that is functionally equivalent to a different protein. 3. A DN comprising the nucleotide sequence of SEQ ID NO: 1.
DNA that hybridizes with A under stringent conditions
And a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 2. (4) a DNA encoding any one of the proteins according to (1), (2) and (3); 5. The DNA according to claim 4, which comprises the coding region of the nucleotide sequence set forth in SEQ ID NO: 1. 6. A vector into which the DNA according to claim 4 or 5 has been inserted. 7. The vector according to claim 6, which is used for gene therapy. 8. A transformant carrying the vector according to claim 6. 9. A method for producing any one of the proteins according to claim 1, comprising a step of culturing the transformant according to claim 8, and recovering an expression product. . 10. A DN which hybridizes with any one of the DNAs according to claim 4 or 5, or a complementary strand thereof.
A, wherein the DNA has a chain length of at least 14 nucleotides. 11. An antisense DN against any one of the DNAs according to claim 4 or 5, or a part thereof.
A. (12) An antibody against any of the proteins according to (1), (2) and (3). 13. The antibody according to claim 12, wherein the antibody is a monoclonal antibody. 14. The antibody according to claim 12, wherein the antibody is at least partially humanized. 15. The protein detection reagent according to claim 1, which comprises the antibody according to any one of claims 12, 13, and 14. A hybridoma that produces the monoclonal antibody according to claim 13. 17. The test compound according to claim 1, comprising the following steps:
2. The method for detecting an activity of binding to a protein according to the above. a) a step of bringing a test compound into contact with the protein of claim 1 b) a step of detecting binding between the protein and the test compound 18. A method for screening a compound having an activity of binding to a protein according to claim 1, comprising a step of selecting a test compound in which the activity of binding to the protein has been detected by the method of claim 17. . 19. The test compound according to claim 1, comprising the following steps:
A method for detecting an activity as a ligand for the protein described in 1. a) a step of bringing a candidate compound for a ligand into contact with a cell that expresses the protein of claim 1 b) a step of detecting uptake by endocytosis via the protein 20. The method for screening a protein ligand according to claim 1, comprising a step of selecting a compound in which the activity of the protein as a ligand is detected by the method according to claim 19. 21. The test compound according to claim 1, which comprises the following steps:
2. A method for detecting an activity of inhibiting the binding between the protein of claim 1 and a ligand thereof. a) a step of contacting the protein according to claim 1 with a ligand in the presence of a test compound, or a ′) a step of contacting the protein according to claim 1 with a test compound before contacting the ligand, 22. The protein according to claim 1, comprising a step of selecting a compound in which the activity of inhibiting the binding of the protein according to claim 1 to its ligand has been detected by the method according to claim 21. For screening for a compound that inhibits the binding between a compound and its ligand. (23) A compound obtainable by the method according to any one of (18), (20) and (22). 24. Use of a compound obtainable by the screening method according to claim 20 or 22 as an inhibitor of cancer metastasis. (25) A method for screening a compound useful as an inhibitor of cancer metastasis by the screening method according to (20) or (22). 26. A transgenic non-human vertebrate in which the expression of a protein encoded by the DNA according to claim 4 has been modified or the modification can be induced. 27. The transgenic non-human vertebra according to claim 26, wherein the expression of the protein encoded by the DNA according to claim 4 is suppressed or the knockout animal is capable of inducing the suppression. animal. 28. The transgenic non-human vertebrate according to claim 26, wherein the non-human vertebrate is a mouse. 29. The DN according to claim 4, comprising the following steps:
A method for producing a transgenic non-human vertebrate in which the expression of a protein encoded by A has been modified or capable of inducing the modification. (A) a step of introducing a vector having a DNA sequence according to claim 4, a DNA sequence derived from a genome encoding the DNA, or a partial DNA sequence thereof, into a fertilized egg or embryonic stem cell of a vertebrate; A) generating an individual from a fertilized egg or embryonic stem cell of step (a); and (c) introducing the DN.
A step of selecting an individual holding A;